The effect of cytokines on the replication of T. gondii within rat retinal vascular endothelial cells.

Toxoplasma gondii infection of the eye can result in a recurrent necrotising retinochoroiditis (TR) which may lead to a permanent loss of visual acuity. The mechanisms responsible for the control of TR within the retina are unknown. The aim of this study was to examine the effects of cytokines on the replication of T. gondii RH strain tachyzoites within rat retinal vascular endothelial (rRVE) cells. Pretreatment of rRVE with IFNgamma, TNF or IL-1beta resulted in a significant decrease in T. gondii replication from day 2 onwards. There was no significant difference in nitric oxide (NO) production by IFNgamma, TNF or IL-1beta treated rRVE as compared to controls at any time point. By comparison, the addition of L-tryptophan to IFNgamma treated cultures significantly restored T. gondii replication from 48 h post inoculation. Thus, IFNgamma, TNF and IL-1beta can significantly inhibit the replication of T. gondii within rRVE. However, this inhibition appears to be independent of NO production. L-tryptophan catabolism may have a role in IFNgamma mediated inhibition of T. gondii replication in rRVE cells.

N-acetylcysteine blocks apoptosis induced by N-alpha-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells.

The serine protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-kappaB activation in T. parva-transformed T-cells, with phosphorylation of IkappaBalpha and IkappaBbeta being inhibited with different kinetics. TPCK-mediated inhibition of IkappaB phosphorylation, NF-kappaB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kappaB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kappaB activation pathway(s).

An immortalized hamster corneal epithelial cell line for studies of the pathogenesis of Acanthamoeba keratitis.

PURPOSE: The protozoan Acanthamoeba produces a severe keratitis in a small percentage of people, especially contact lens-wearers. The purpose of this work was to develop and characterize an immortalized line of hamster corneal epithelial cells to be used in studies of the pathogenesis of Acanthamoeba keratitis. METHODS: Hamster corneal epithelial cells were maintained in primary culture and immortalized using simian virus 40 (SV40). Foci of transformed cells were cloned and subsequently characterized by phase-contrast microscopy and immunocytochemistry. Growth characteristics of the clone that were analyzed included loss of dependence on conditioned medium and ability to grow in soft agar. Cytotoxicity experiments were performed, to determine whether the selected clone was susceptible to Acanthamoeba infection in vitro. RESULTS: A cell line which exhibited epithelial morphology, as determined by phase contrast microscopy, was selected and cloned. Immunocytochemistry demonstrated the presence of keratin in the cloned cells, confirming the epithelial nature of the cell line. Immortalization was shown by loss of dependence on fibroblast-conditioned medium, ability to form colonies in soft agar and no apparent senescence following numerous passages in culture. This cell line was found to be sensitive to the cytotoxic effects of a pathogenic strain of Acanthamoeba. CONCLUSIONS: An immortalized line of hamster corneal epithelial cells was developed. This clone is susceptible to infection with Acanthamoeba and will be a useful tool with which to investigate the pathogenesis of Acanthamoeba keratitis.