Comparative efficacy of antiviral drugs on human ocular fibroblasts.

The effects of several antiviral drugs on fibroblast attachment and proliferation from human Tenon's capsule were investigated. These drugs included purine nucleoside analogs, vidarabine and acyclovir (ACV); pyrimidine nucleoside analog, AZT; and a synthetic cyclic primary amine, amantadine. Fibroblast attachment and proliferation inhibition were determined by Coulter counter, a colorimetric assay of the enzyme hexosaminidase, and a 3H-thymidine uptake assay. Amantadine and AZT inhibited fibroblast attachment at concentrations higher than 6.61 x 10(-4)M and 3.73 x 10(-4) M, respectively. Amantadine and AZT had inhibitory effects on fibroblast proliferation as early as day 1, whereas vidarabine and ACV manifested their inhibitory effects after day three by Coulter counter and hexosaminidase assays. For amantadine, AZT, ACV and vidarabine, the 50% inhibitory dose (ID50) were 4.94 x 10(-5) M, 1.26 x 10(-5) M, 4.60 x 10(-4) M, and 1.52 x 10(-5) M at day 9, respectively, as measured by 3H-thymidine uptake assay. All four antiviral agents tested had inhibitory effects on human ocular fibroblast proliferation and their inhibitory potential decreased in the order of amantadine > or = vidarabine > AZT > or = ACV.

Effects of timolol, betaxolol, and levobunolol on human tenon's fibroblasts in tissue culture.

Evidence has been found suggesting that long-term therapy with topical antiglaucoma medications may decrease the success of glaucoma filtering surgery. To investigate this question further, the antiproliferative effects of the preservative benzalkonium chloride and three pure and commercially available beta-adrenergic antagonist preparations (timolol, betaxolol, and levobunolol) were studied on tissue cultures of human Tenon's capsule fibroblasts. Each drug preparation was tested on three different cell lines. Fibroblast growth was measured with tritiated thymidine uptake and hexosaminidase assays. Trypan blue uptake was used to assess cell viability microscopically. The commercially available preparations containing benzalkonium chloride and those of betaxolol and levobunolol without the preservative had similar inhibitory doses for 50% of cells. The timolol preparation without preservative was significantly less toxic than its commercially available one. The three tested beta-adrenergic blockers did not stimulate fibroblast proliferation directly in this in vitro model. Even when the cultures were washed free of the drugs, growth continued to be suppressed, suggesting that the inhibition was not reversible. An increase in fibroblasts and inflammatory cells after long-term antiglaucoma medical therapy thus may be caused not by a direct stimulation of cell proliferation but by chronic inflammation from the irritating effects of antiglaucoma medications and/or their preservatives.

The effects of the fluorinated pyrimidines FUR, FUdR, FUMP, and FdUMP on human Tenon's fibroblasts.

5-Fluorouracil (5-FU) has effectively inhibited fibroblast proliferation to prevent scar formation and bleb failure after glaucoma filtering surgery. To identify more potent but less toxic antiproliferative drugs, the authors studied cell attachment and proliferation of 5-FU metabolites: 5-fluorouridine (FUR), 5-fluorodeoxyuridine (FUdR), 5-fluorouridine-5'-monophosphate (FUMP), and 5-fluorodeoxyuridine-5'-monophosphate (FdUMP) on human Tenon's fibroblasts in vitro.

Characterization of human and rabbit pigmented and nonpigmented ciliary body epithelium.

Nonpigmented epithelial (NPE) and pigmented epithelial (PE) cells were carefully dissected from both human and rabbit ciliary processes and have been maintained in vitro and partially characterized by morphology and immunocytochemical techniques using polyclonal and monoclonal antibodies against S-100 proteins, collagen type I and type III. The tissue distribution of these proteins was studied in formalin fixed deparaffinized tissue sections of human and rabbit eyes by immunoperoxidase staining techniques. Both NPE and PE cell lines from human and rabbit showed hexagonal morphology by light microscopy; distinct granules containing pigment could be visualized in the PE cell lines, but not in the NPE cells. Antibodies against S-100 proteins stained NPE layer intensely and PE layer slightly in the human tissue sections. The staining was less intense in rabbit tissues than human tissues. The ciliary body stroma was positive for collagen type III and negative for collagen type I or S-100.

Inhibition of rabbit ocular fibroblast proliferation by 5-fluorouracil and cytosine arabinoside.

Inhibition of rabbit subconjunctival fibroblast attachment and proliferation by the antimetabolites 5-fluorouracil (5-FU) and cytosine arabinoside (ara-C) was determined by radionucleotide uptake, cell counting, and colorimetric assays for the concentration range of 1000 to 0.0001 micrograms/ml over an 11-day period. The mean 50% inhibitory doses (ID50s) against proliferation were calculated for each assay. Rabbit fibroblast attachment was not inhibited at any drug concentration by either 5-FU or ara-C. Ara-C was a 10 to 100 times more potent inhibitor of rabbit fibroblast proliferation than 5-FU. The mean ID50s for rabbit subconjunctival fibroblasts were compared with the mean ID50s from a similar series of experiments conducted in our laboratory on human subconjunctival fibroblasts. Unpaired t-test analysis showed a significant difference between the inhibitory effects of 5-FU on rabbit and human fibroblast proliferation. An ID50 against rabbit fibroblasts was detectable after 24 hours of incubation with 5-FU by the 3H-thymidine uptake assay, whereas the ID50 against human fibroblasts was detectable after 48 hours of incubation. Once inhibition of proliferation occurred, however, human fibroblasts were up to six times more sensitive to the antiproliferative effects of 5-FU than rabbit fibroblasts as measured by the 3H-thymidine uptake assay (p = 0.0005). Unpaired t-test analysis showed no statistical difference between the ID50s of ara-C on rabbit and human fibroblasts. Starting on day 3, however, doses greater than 1 micrograms/ml of ara-C were cytotoxic to rabbit fibroblasts but only cytostatic to human fibroblasts as determined by trypan blue uptake assay microscopically. Rabbit ocular fibroblasts may be useful in modelling the proliferation of human ocular fibroblasts in vitro to a limited degree. This tissue culture system may be useful for predicting optimal drug dosages for in vivo rabbit and human glaucoma filtering surgery.

The effects of 5-fluorouridine, 5-fluorodeoxyuridine, and 5-fluorodeoxyuridine monophosphate on rabbit tenon's capsule fibroblasts in vitro.

Inhibition of rabbit subconjunctival fibroblast attachment and proliferation by 5-fluorouridine (FUR), 5-fluoro-2 deoxyuridine (FUdR), and 5-fluoro-2-deoxyuridine-5-monophosphate (FdUMP), was determined by 3H-adenosine uptake, cell counting, and colorimetric assays for the concentration range of 1000 to 0.0001 micrograms/ml over an 9 day period. The mean 50% inhibitory doses against proliferation were calculated for each assay. Rabbit fibroblast attachment was not inhibited at any drug concentration by either FUR, FUdR, or FdUMP. For rabbit fibroblast proliferation, FUR was found to be 10-100 fold more potent than FUdR and FdUMP. When comparing the human and rabbit cells, the unpaired t-test analysis showed no consistent statistical difference of the ID50s for FUR, FUdR or FdUMP. Rabbit ocular fibroblasts may be useful in modeling the proliferation of human ocular fibroblasts. These in vitro results may be useful for predicting optimal drug dosages for future in vivo testing of these drugs.

The effect of 5-fluorouracil and cytarabine on human fibroblasts from Tenon's capsule.

The in vitro cellular inhibitory effects of two pyrimidine antimetabolites, 5-fluorouracil (5-FU) and cytarabine (ara-C), on the attachment and proliferation of human Tenon's capsule fibroblasts after 1, 2, 3, 5, 7, and 10 days of growth were measured with a Coulter counter, a colorimetric method using the endogenous enzyme hexosaminidase, and 3H-thymidine uptake. Neither 5-FU nor ara-C affected cell attachment. The 50% inhibitory dose (ID50) for 5-FU, as measured by the Coulter counter and hexosaminidase assay, was 0.2 and 0.4 micrograms/ml, respectively, at day 5 and decreased to 0.01 and 0.10 micrograms/ml, respectively, on later days. The ID50 for ara-C as measured by the Coulter counter and hexosaminidase assay was 0.01 and 0.1 micrograms/ml at day 3 and remained constant over time. Much lower ID50s were measured by thymidine uptake for both drugs. These findings may indicate that 5-FU has a delayed effect on cellular proliferation due to conversion into more active metabolites. The ara-C has a direct and constant inhibitory effect on cellular proliferation and is ten times more potent than 5-FU as an antiproliferative drug. Thus ara-C may have clinical utility in preventing failure of glaucoma filtering surgery.