Laser-induced fluorescence during photorefractive keratectomy: a method for controlling epithelial removal.

PURPOSE: Laser-induced fluorescence is generated during ablation of corneal tissue with the argon-fluoride 193-nm excimer laser. To investigate possible changes in laser-induced fluorescence spectra emitted during the transition between epithelium and stroma, we developed a system using an intensified charge-coupled device to achieve fast per-pulse temporal resolution of laser-induced fluorescence. METHODS: Freshly enucleated human cadaver eyes were subjected to 193-nm excimer laser keratectomy. During the procedure, laser-induced fluorescence was measured using an intensified charge-coupled device. Changes in laser-induced fluorescence were detected and used to control epithelial removal. Depth of ablation was determined histologically. RESULTS: The 193-nm excimer laser pulses induced both visible and ultraviolet fluorescence from corneal epithelium and stroma. In each layer two peaks predominated, one at 405 nm and the other at 346 nm. There was a rapid threefold reduction in the 346-nm ultraviolet peak at the transition from epithelium to stroma. CONCLUSIONS: By monitoring changes in laser-induced fluorescence at the epithelial-stromal interface, the clinician may be able to control corneal epithelial removal more precisely and reproducibly before performing photorefractive ablation.

Biomarkers of marine pollution observed in species of mullet living in two eastern Mediterranean harbours.

The activities of enzymes associated with xenobiotic metabolism and or oxidative processes, and the levels of aromatic DNA adducts, have been determined in the livers of grey mullet Oedalechilus labeo and Lisa ramada living in two eastern Mediterranean harbours. Glutathione peroxidase GSH P activity was 2.5 times higher 9 IU g-1 liver and glutathione reductase GSSG R activity was twice as high 2.5 IU g-1 liver in fish from the more polluted harbour at Mersin than in the harbour near Erdemli. Superoxide dismutase SOD activity was 25 lower 4.3 IU g-1 liver in the more polluted harbour. The concentrations of glutathione and malondialdehyde varied both with species and environment by a factor of 2.5-3. DNA adducts in liver were determined by 32P postlabelling. In Oedalechilus labeo in the more polluted harbour, adduct levels were 258 21 adducts per 108 nucleotides mean SE; two groups of Lisa ramada were distinguished having 261 48 and 30 6 adducts per 108 nucleotides, respectively. The average adduct level in a group of mullet of mixed species in the less polluted harbour was 3.3 2.3 adducts per 108 nucleotides. The results illuminate the ability of mullet to live in contaminated marine environments, and show that enzyme activities and liver DNA adduct levels can serve as indicators of marine pollution.

Effect of diclofenac, ketorolac, and fluorometholone on arachidonic acid metabolites following excimer laser corneal surgery.

OBJECTIVE: To compare the ability of several topical anti-inflammatory agents to modulate the production of prostaglandin E2 after excimer laser ablation in rabbit cornea. METHODS: Adult New Zealand white rabbits were subjected to phototherapeutic keratectomy with a commercially available excimer laser. Prostaglandin E2 and leukotriene B4 were detected by enzyme-linked immunoassay, and leukocyte infiltration was determined histologically. RESULTS: Prostaglandin E2 and leukocyte infiltration increased in the cornea after excimer ablation. Treatment with topical fluorometholone and diclofenac sodium significantly reduced prostaglandin E2 levels. Corneas treated with diclofenac had significantly higher levels of leukocyte infiltration than those treated with ketorolac tromethamine. No changes in leukotriene B4 levels were detected in this model. CONCLUSIONS: As a group, topical anti-inflammatory medications tend to lower prostaglandin E2 levels in rabbit corneas subjected to excimer ablation, but differ in their ability to reduce polymorphonuclear leukocyte infiltration. Further work is needed in this model to understand how these drugs alter leukocyte infiltration of the remaining stromal bed.

Arachidonic acid metabolites after excimer laser corneal surgery.

OBJECTIVE: We investigated early mediators of inflammation following excimer laser ablation in a rabbit cornea model. The ability of topical anti-inflammatory agents to influence these responses was also examined. METHODS: Adult New Zealand white rabbits were subjected to photorefractive keratectomy with a 193-nm argon fluoride excimer laser. Prostaglandin E2 and leukotriene B4 levels were measured using an enzyme immunoassay, and leukocyte infiltration was determined histologically. RESULTS: Prostaglandin E2 production was rapid and sustained, but we were unable to detect the presence of leukotriene B4. Relative to control, postoperative topical diclofenac sodium treatment caused a significant decrease in prostaglandin E2 levels and a significant increase in corneal leukocytes at 10 hours. Fluorometholone treatment did not significantly alter prostaglandin E2 levels but markedly depressed leukocyte ingress. CONCLUSIONS: Diclofenac reduces prostaglandin E2 levels but not leukocyte infiltration in the cornea following photorefractive keratectomy and thus may be useful clinically to reduce postsurgical pain.

Characterization of the T cell antigen receptor--p60fyn protein tyrosine kinase association by chemical cross-linking.

Engagement of the TCR by specific antigen results in activation of a tyrosine kinase pathway. A candidate for the kinase responsible for the rapid tyrosine phosphorylation detected with T cell activation is p60fyn, a member of the src kinase family. In an earlier study [Samelson et al. (1990) Proc. Natl Acad. Sci. USA 87:4358] this enzyme was co-immunoprecipitated with the TCR from T cells solubilized in digitonin. In that study a sensitive in vitro kinase assay was used to detect the associated p60fyn. It was subsequently found that the reproducibility of the interaction depended on lot-to-lot variations in digitonin. To eliminate the possibility that the association of antigen receptor and kinase is an artifact of solubilization with ill-defined digitonin preparations, a cross-linking protocol was developed to stabilize the interaction between the TCR and p60fyn. T cells were permeabilized with tetanolysin and proteins were cross-linked with the water soluble chemical cross-linker, 3,3' dithiobis(sulfosuccinimidylpropionate). These experiments allowed the confirmation of the interaction between the TCR, p60fyn, and several additional proteins. The cross-linking studies also enabled the mapping of the interaction of p60fyn and associated proteins to the TCR zeta-chain. This technique should have a general use in stabilizing interactions between other receptors and molecules required for intracellular signaling.

Multiple signal transduction pathways activated through the T cell receptor for antigen.

The T cell receptor for antigen (TCR) is a multichain complex on the surface of T lymphocytes which binds peptide antigen and transduces a transmembrane signal leading to IL-2 secretion. Engagement of the TCR leads to activation of a tyrosine phosphorylation pathway and a phospholipase C (PLC) pathway leading to activation of protein kinase C (PCK). Currently available data suggest that the primary event in signal transduction is tyrosine kinase activation, since when this pathway is inhibited, PLC activation is blocked and there is no production of IL-2. The nature of the tyrosine kinase which initiates the signaling cascade is currently unknown. The CD4/CD8 associated kinase p56lck clearly plays a role in tyrosine phosphorylation, but it is clearly not the only tyrosine kinase involved. Studies demonstrating physical association of p59lyn with the TCR implicate fyn as an important candidate for the TCR tyrosine kinase. The protein tyrosine phosphatase CD45 also plays a critical early role in signal transduction since in cells where it is deficient, neither tyrosine kinase activation nor later signaling events are seen. The importance of the PLC/PKC pathway is illustrated by the fact that activation of this pathway alone may lead to IL-2 production. However, there may also be other mechanisms which can generate an IL-2 response. Two proteins known to be involved in growth regulation--p21ras and c-raf--have now been shown to be downstream targets of the PLC/PKC pathway.

Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction.

The binding of antigen to the multicomponent T-cell receptor (TCR) activates several signal transduction pathways via coupling mechanisms that are poorly understood. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC). TCR activation by antigen, lectins, or anti-TCR monoclonal antibody has also been shown to cause increases in tyrosine phosphorylation of TCR-zeta and other substrates, suggesting stimulation of protein tyrosine kinase (PTK) activity. A critical question is whether these two pathways, PLC and PTK, are independently activated or whether one initiates and/or regulates the other. In the former case, PLC activation could be coupled to the TCR via a GTP-binding protein (G protein). We have reported, however, that tyrosine phosphorylation of intracellular substrates precedes detection of PLC activation and intracellular calcium elevation, suggesting that inositol phospholipid turnover in T cells is initiated by a PTK pathway. In this study, we test this hypothesis by treating T cells with the drug herbimycin A. We demonstrate that this agent inhibits substrate tyrosine phosphorylation, TCR-mediated inositol phospholipid hydrolysis, and calcium elevation. In contrast, under these conditions G-protein-mediated PLC activity, as tested by addition of aluminum fluoride, remains intact. Furthermore, whereas herbimycin treatment prevents TCR-mediated interleukin 2 production and interleukin 2 receptor expression, phorbol ester-induced effects are substantially resistant to herbimycin. The drug thus appears to abrogate TCR-mediated signaling without affecting distal signaling mechanisms.

Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor.

Activation of the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of the TCR zeta chain and other intracellular substrates. Two other T-cell integral membrane proteins, CD4 and CD8, are associated with the protein-tyrosine kinase (PTK), lck. Despite evidence that activation of this enzyme results in TCR-zeta chain phosphorylation, it has not been shown that the TCR activates lck. We have sought evidence that the TCR is associated with a PTK. In this study we use digitonin to solubilize a murine T-cell hybridoma and demonstrate that antibodies binding extracellular but not intracellular domains of the TCR specifically coprecipitate only the fyn PTK and not lck or yes, two other kinases found in these cells. The association of the fyn PTK with the TCR might enable the T cell to independently regulate two PTKs through surface receptors.