Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome—with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)—but very limited changes in the small RNA transcriptome—totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis

The synthesis and degradation of collagenase-degradable poly(2-hydroxyethyl methacrylate)-based hydrogels and sponges for potential applications as scaffolds in tissue engineering

A collagenase-cleavable peptide-based crosslinking agent was synthesized and was incorporated into PHEMA sponges, and P[HEMA-co-MeO-PEGMA] gels and sponges [HEMA 2-hydroxyethyl methacrylate, PHEMA = poly(2-hydroxyethyl methacrylate), MeO-PEGMA = poly(ethylene glycol) monomethyl ether methacrylate]. PHEMA and P[HEMA-co-MeO-PEGMA] sponges had polymer droplet morphologies where the dimensions of the morphological features were three to five times larger compared to sponges that were crosslinked with tetraethylene glycol dimethacrylate (TEGDMA), while the P[HEMA-co-MeO-PEGMA] gels had similar morphologies regardless of the crosslinking agent. The differences in the dimensions of the morphologies of the sponges were attributed to differences in hydrophilicities of the crosslinking agent. When incubated in a collagenase solution, PHEMA sponges did not degrade, but P[HEMA-co-MeO-PEGMA] gels took 28 days to degrade and the P[HEMA-co-MeO-PEGMA] sponges took 101 days to degrade to 8% dry weight remaining. A cytotoxicity assay showed that the hydrogels do not elicit any cytotoxic response in vitro

Effect of the sterilization method on the properties of Bombyx mori silk fibroin films

We have compared the effects of different sterilization techniques on the properties of Bombyx mori silk fibroin thin films with the view to subsequent use for corneal tissue engineering. The transparency, tensile properties, corneal epithelial cell attachment and degradation of the films were used to evaluate the suitability of certain sterilization techniques including gamma-irradiation (in air or nitrogen), steam treatment and immersion in aqueous ethanol. The investigations showed that gamma-irradiation, performed either in air or in a nitrogen atmosphere, did not significantly alter the properties of films. The films sterilized by gamma-irradiation or by immersion in ethanol had a transparency greater than 98% and tensile properties comparable to human cornea and amniotic membrane, the materials of choice in the reconstruction of ocular surface. Although steam-sterilization produced stronger, stiffer films, they were less transparent, and cell attachment was affected by the variable topography of these films. It was concluded that gamma-irradiation should be considered to be the most suitable method for the sterilization of silk fibroin films, however, the treatment with ethanol is also an acceptable method

Improving the cellular invasion into PHEMA sponges by incorporation of the RGD peptide ligand: The use of copolymerization as a means to functionalize PHEMA sponges

Amonomer that contained the RGD ligandmotifwas synthesized and copolymerized with 2-hydroxyethylmethacrylate using polymerization-induced phase separation methods to form poly(2-hydroxyethyl methacrylate)-based hydrogel sponges. The sponges had morphologies of aggregated polymer droplets and interconnected pores, the pores having dimensions in the order of 10 μm typical of PHEMA sponges. RGD-containing moieties appeared to be evenly distributed through the polymer droplets. Compared to PHEMA sponges that were not functionalizedwith RGD, the new sponges containing RGD allowed greater invasion by human corneal epithelial cells, by advancing the attachment of cells to the surface of the polymer droplets