RESUMEN

Purpose/Aim: Corneal abrasions and nonhealing corneal epithelial defects are common conditions that cause pain and sometimes are slow to heal. Histatins, a family of histidine-rich peptides, have been implicated in oral and skin epithelial wound healing, and have been shown to be effective in vitro in human corneal epithelial cells. The objective of this study was to test the efficacy of histatin-1 on corneal epithelial wound healing in rabbits. MATERIALS & METHODS: Twenty-two (22) rabbits were separated into four treatment groups, each containing 3-7 rabbits. Treatments included three histatin-1 formulations (0.1 ug/ml. 1 ug/ml, and 10 ug/ml) and one inactive vehicle, one drop given three times per day. Eight (8) mm circular wounds were created using 0.5 ml of 20% ethyl alcohol in the right eye of each rabbit. A masked observer photographed each eye twice daily using slit-lamp biomicrophotography. Wound area was analyzed by using ImageJ. Statistical analysis was conducted using Graphpad Prism. RESULTS: Wound recovery was faster in animals given 0.1 ug/ml, 1 ug/ml, and 10 ug/ml when compared to the vehicle solution at 6, 24, and 30 hours after wound creation (p < 0.01). No adverse events were observed in any eyes. When analyzing area under the curve, % recovered area was higher overall in the 0.1 ug/ml (p < 0.01), 1 ug/ml (p < 0.01), and 10 ug/ml (p < 0.001) groups when compared to the vehicle solution. Hourly healing rate was also observed to be faster in the 0.1 ug/ml, 1 ug/ml, and 10 ug/ml groups (p < 0.001) at 24 hours postinjury suggesting an accelerated healing process as compared to the vehicle group. CONCLUSION: This study represents the first in vivo experiment evaluating and confirming the efficacy of topical histatin on the corneal epithelium wound healing. Further studiesare warranted to better understand the mechanism and safety of topical histatin-1 in corneal epithelial wound-healing and its potential role for human disease treatment.

Asunto(s)

RESUMEN

PURPOSE: To determine the changes in dry eye disease (DED) severity and the percentage of cells expressing HLA-DR on the ocular surface following treatment with lubricant eyedrops containing polyethylene glycol and propylene glycol (PEG/PG) and the gelling agent hydroxypropyl guar (HP-Guar). PATIENTS AND METHODS: Nineteen patients with DED used PEG/PG + HP-Guar eyedrops four times per day for 30 days. Assessments included DED severity (Ocular Surface Disease Index [OSDI], corneal staining, conjunctival staining, tear film break-up time [TFBUT], and Schirmer testing) and impression cytology of the conjunctiva with masked flow cytometry at baseline and at 30 days. RESULTS: There was a significant decrease in corneal staining (P<0.01), OSDI (P=0.02), and TFBUT (P<0.01) following treatment with PEG/PG + HP-Guar. Results from flow cytometry revealed a significant decrease in cells expressing HLA-DR (P=0.02). CONCLUSION: Treatment with PEG/PG + HP-Guar eyedrops showed improvement in dry eye severity and reduction in surface inflammation as indicated by a reduction in HLA-DR expression.

RESUMEN

There are currently no validated minimally invasive objective metrics for the classification and evaluation of ocular surface diseases and/or for evaluating treatment efficacy. We thus sought to establish a standardized methodology for determining the relative amount of the inflammatory biomarker HLA-DR on the ocular surface and to evaluate the precision, reliability and repeatability of its use for large multicenter clinical trials and translational research studies of ocular surface disease. Multiple studies were conducted to establish a Standard Operating Procedure (SOP) for utilizing HLA-DR expression as a minimally invasive, objective, ocular surface inflammatory biomarker. The established SOPs provide specific guidelines for HLA-DR collection and analysis, in order to incorporate it reliably into multicenter clinical trials and/or translational research. Duplicate cell samples from impression cytology (IC) samples of both normal and dry eye individuals were collected and split to assess repeatability (between the splits and between the duplicate samples). To determine storage capability, one duplicate was stained immediately and the other after 30 days cold storage. To demonstrate the feasibility of the use of the SOP for a multicenter clinical trial, clinicians out-of-state were trained to collect IC samples, and the samples shipped to our Biomarker Laboratory, logged, processed and analyzed. Demonstration of the ability to incorporate of IC into a randomized double masked clinical trial of dry eye disease (DED) was performed. In all cases, processing and analyses were performed by a masked independent observer. The validity/viability of the SOPs was established by demonstrating that: 1) sufficient numbers of cells can be collected via IC; 2) the precision/repeatability of the relative biomarker expression quantified in samples; 3) personnel at distant sites can be taught to collect, store and ship samples successfully; 4) samples can be stored for up to 30 days (refrigeration) before processing without affecting results; 5) IC can be incorporated into a double blind randomized clinical trial (RCT) of DED; and 6) the Biomarker Laboratory can track a large number of masked samples reliably. In conclusion, our standard operating procedure for impression cytology analysis of HLA-DR expression appears to be repeatable and reproducible for use in multicenter clinical trials, providing a minimally invasive objective biomarker of inflammation of the ocular surface.

Asunto(s)

RESUMEN

PURPOSE: sPLA2-IIa is a biomarker for many inflammatory diseases in humans and is found at high levels in human tears. However, its role in ocular surface inflammation remains unclear. An experimentally induced BALB/c mouse dry eye (DE) model was used to elucidate the role of sPLA2-IIa in ocular surface inflammation. METHODS: BALB/c mice were subcutaneously injected with scopolamine and placed in a daytime air-drying device for 5 to 10 days. Control mice received no treatment. DE status was evaluated with tear production with a phenol-red thread method. Tear inflammatory cytokines were quantified by multiplex immunoassays. Ocular surface inflammation and sPLA2-IIa expression were examined by immune-staining and quantitative (q)RT(2)-PCR. Conjunctiva (CNJ) of the mice was cultured for prostaglandin E2 production induced by sPLA2-IIa with various amount of sPLA2-IIa inhibitor, S-3319. RESULTS: Treated mice produced fewer tears and heavier corneal (CN) fluorescein staining than the untreated controls (P < 0.001). They also revealed lower goblet cell density (P < 0.001) with greater inflammatory cell infiltration within the conjunctiva, and higher concentration of tear inflammatory cytokines than the controls. Moreover, treated mice showed heavier sPLA2-IIa immune staining than the controls in the CNJ epithelium, but not in the CN epithelium or the lacrimal gland. Treated mice exhibited upregulated sPLA2-IIa and cytokine gene transcription. Furthermore, CNJ cultures treated with sPLA2-IIa inhibitor showed significantly reduced sPLA2-IIa-induced inflammation. CONCLUSIONS: This is the first report regarding sPLA2-IIa in the regulation of ocular surface inflammation. The findings may therefore lead to new therapeutic strategies for ocular surface inflammation, such as DE disease.

Asunto(s)

RESUMEN

PURPOSE: Most eye drops contain preservatives; benzalkonium chloride (BAK) is most common. Recent data demonstrated BAK adding to toxicity. BAK is degraded into hydrogen peroxide (H(2)O(2)), which in even small amounts is known to be an irritant. Increased toxicity should cause localized inflammation with increased elaboration of inflammatory biomarkers. To evaluate the inflammation BAK causes to the ocular surface, enzyme linked immunosorbant assays (ELISAs) were utilized to quantify the levels of inflammatory biomarkers in response to BAK and/or H(2)O(2). METHODS: Immortalized human conjunctival and corneal epithelial cells were exposed to: BAK (0.001%-0.1%), hydrogen peroxide (H(2)O(2)) (0.01%-0.1%), and cell media for 1 h. Cytokine quantification was performed via enzyme-linked immunosorbent assays [ELISAs]). Additional experimentation was performed in which testing solutions were replaced with media after 1 h and the resulting supernatants quantified after 24 h. RESULTS: BAK induced significant amounts of interleukin (IL-) 1 and tumor necrosis factor (TNF), but only moderate amounts of C-reactive protein (CRP), IL- 10 and 12, and H(2)O(2). Lower concentrations of BAK induced proportionally less elaboration. Replacing the test solutions with media and providing 23 h for cytokine elaboration significantly increased TNF, but not IL-1. Lipopolysaccharide (LPS) positive controls induced substantial elaboration/release of both IL-1 and TNF as did in increasing the exposure to the full 24 h. CONCLUSIONS: After 1 h of exposure, BAK increased quantities of all biomarkers. The biomarkers in decreasing order of induction/upregulation were: TNF > or = IL-1 > or = IL-12 > or = IL-10 > or = CRP. Even low concentrations caused some degree of inflammation. Replacing the testing solution with media and providing 23 h for cytokine elaboration, significantly increased the elaboration/release of TNF, but not IL-1, as compared to the 1-h BAK exposure. Whereas increasing the exposure to the full 24 h by not removing the testing solution at the 1-h time point significantly increased the elaboration/release of both IL-1 and TNF.

Asunto(s)

RESUMEN

PURPOSE: Nearly all eye drops contain preservatives to decrease contamination. Nonpreservatives such as disodium-ethylene diamine tetra-acetate (EDTA) and phosphate-buffered saline are also regularly added as buffering agents. These components can add to the toxicity of eye drops and cause ocular surface disease. To evaluate the potential toxicity of these common components and their comparative effects on the ocular surface, a tissue culture model utilizing immortalized corneal and conjunctival epithelial cells was utilized. METHODS: Immortalized human conjunctival and corneal epithelial cells were grown. At confluency, medium was replaced with 100 microL of varying concentrations of preservatives: benzalkonium chloride (BAK), methyl paraben (MP), sodium perborate (SP), chlorobutanol (Cbl), and stabilized thimerosal (Thi); varying concentrations of buffer: EDTA; media (viable control); and formalin (dead control). After 1 h, solutions were replaced with 150 microL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide). After 4 h, solutions decanted, 100 microL of acid isopropanol added, and the optical density determined at 572 nm to evaluate cell viability. RESULTS: Conjunctival and corneal cell toxicity was seen with all preservatives. Depending upon concentration, BAK exhibited from 56% to 89% toxicity. In comparison, Cbl exhibited from 50% to 86%, MP from 30% to 76%, SP from 23% to 59%, and Thi from 70% to 95%. EDTA with minimal toxicity (from 6% to 59%) was indistinguishable from SP. CONCLUSIONS: Generally, the order of decreasing toxicity at the most commonly used concentrations: Thi (0.0025%) > BAK (0.025%) > Cbl (0.25%) > MP (0.01%) > SP (0.0025%) approximately EDTA (0.01%). Even at low concentration, these agents will cause some degree of ocular tissue damage.

Asunto(s)

RESUMEN

PURPOSE: To evaluate the toxicity of a variety of the fluoroquinolone antibiotics on the ocular surface by using tissue culture models of corneal epithelial cells and conjunctival epithelial cells. METHODS: Immortalized conjunctival (CCC) and human corneal (HCE) epithelial cells were grown and when confluent the cells allowed to air dry for 1 hour. Medium was then replaced with 100 microL of one of the following: 1) Vigamox [moxifloxacin (0.5%: MX)]; (2) Zymar [gatifloxacin (0.3%: GA)]; 3) Quixin [levofloxacin (0.5%: LE)]; 4) Ocuflox [ofloxacin (0.3%: OF)]; 5) Ciloxan [ciprofloxacin (0.3%: CP)]; 6) medium (viable control); 7) "normal"/physiologic saline; 8) formalin (dead control). After one hour, 150 microL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide was added and incubated for 4 hours. After decanting, precipitate was dissolved in 150 microL of isopropanol. Absorbance was determined at 572 nm. RESULTS: The lowest amount of cell death was associated with the viable control. All ophthalmic preparations showed both corneal and conjunctival cell toxicity. Aside from the viable control, normal saline showed the next lowest amount of toxicity. Of the topical ocular antibiotics tested, MX showed the least amount of toxicity. All of the other antibiotics tested were statistically indistinguishable from each other. CONCLUSIONS: All of the topical ocular antibiotics tested showed evidence of both corneal and conjunctival toxicity (MX < OF < or = LE < or = CP < or = GA), although only MX was statistically significant. Whether this finding reflects on in vivo wound healing remains to be determined. This model provides a rapid and cost-effective method to screen for surface toxicity of topical agents.

Asunto(s)

RESUMEN

PURPOSE: Pseudomonas aeruginosa ocular infections most frequently originate from an environmental source; successful treatment with various ocular antibiotics is well established. However, emergence of resistant clones to available antibiotics poses a real threat to successful treatment. The purpose of this study was to evaluate the antibiotic susceptibilities of 100 random clinical isolates of P. aeruginosa to levofloxacin, moxifloxacin, and gatifloxacin, potential agents for the treatment of ocular infections caused by this microorganism. METHODS: One hundred consecutive strains of P. aeruginosa were isolated from clinical specimens submitted to the clinical microbiology hospital laboratory. Duplicate isolates were not included. The minimum inhibitory concentrations (MICs) of these isolates were determined by using Etests, performed according to the manufacturer's instructions. American Type Culture Collection (ATCC) strains of Escherichia coli, P. aeruginosa, and Staphylococcus aureus served as reference controls. RESULTS: Although most isolates were susceptible to levofloxacin, moxifloxacin, and gatifloxacin and the MICs were not significantly different, significant numbers were resistant. The standardized controls rendered expected MICs. The susceptibility of the isolates varied with regard to source, and resistant strains showed increased resistance. CONCLUSIONS: Based on the data, the treatment of ocular infections caused by P. aeruginosa with levofloxacin, moxifloxacin, and gatifloxacin still has a high likelihood of success. However, six of the isolates collected were resistant to all three of the fluoroquinolones tested. Based on the data, clinicians must be aware that clinical resistance can occur even with the newer fluoroquinolones.

Asunto(s)

RESUMEN

BACKGROUND: Adenovirus (Ad), associated with significant morbidity, has no topical treatment. A leading CTC compound (CTC-96), a Co(III) chelate, was found to have potent in vitro and in vivo antiviral efficacy against herpes viruses. In this study CTC-96 is being tested for possible anti-Adenovirus activity. METHODS: The biological anti-adenovirus activity of CTC-96 in concentrations from 5 to 250 ug/ml, was evaluated initially by viral inactivation (viral exposure to CTC-96 followed by dilution and inoculation of cells), virucidal (viral exposure to CTC-96 and inoculation of cells without dilution) and antiviral (effect of CTC-96 on previously adsorbed virus) plaque assays on HeLa (human cervical carcinoma), A549 (human lung carcinoma) and SIRC (rabbit corneal) cells. After verifying the antiviral activity, New Zealand White rabbits were infected with Ad-5 into: 1) the anterior cul-de-sac scarifying the conjunctiva (Group "C+"); 2) the anterior cul-de-sac scarifying the conjunctiva and cornea (Group "CC+"); 3) the stroma (Group "CI+"). Controls were sham-infected ("C-", "CC-", "CI-"). Other rabbits, after "CC", were treated for 21 days with: 1) placebo, 9x/day ("-"); 2) CTC-96, 50 ug/ml, 9x/day ("50/9"); CTC-96, 50 ug/ml, 6x/day ("50/6"); CTC-96, 25 ug/ml, 6x/day ("25/6"). All animals were monitored via examination and plaque assays. RESULTS: In vitro viral inactivation, virucidal and antiviral assays all demonstrated CTC-96 to be effective against Adenvirus type 5 (ad-5). The in vivo model of Ad keratoconjunctivitis most similar to human disease and producing highest viral yield was "CC". All eyes (6/6) developed acute conjunctivitis. "CI" yielded more stromal involvement (1/6) and iritis (5/6), but lower clinical scores (area x severity). Infection via "C" was inconsistent (4/6). Fifty (50) ug/ml was effective against Ad-5 at 6x, 9x dosings while 25 ug/ml (6x) was only marginally effective. CONCLUSION: CTC-96 demonstrated virucidal activity against Ad5 in tissue culture with HeLa, A549 and SIRC cell lines. Animal Model Development: 1) "CC" produced conjunctival infection with occasional keratitis similar to human disease; "CI" yielded primarily stromal involvement; 2) "C" consistently produced neither conjunctivitis nor keratitis.CTC Testing: 1) Conjunctivitis in all eyes; 2) Resolution fastest in "50/9" ("50/9". "50/6" > "25/6" > "-"); 3) Efficacy in "50/6" was not statistically different than "50/9"; 4) Conjunctival severity was lower in treatment groups then controls; 5) Little corneal or intra-ocular changes were noted.

Asunto(s)

RESUMEN

PURPOSE: Because stem cells exhibit high self-renewal capacity, slow cycling, and high proliferative potential, and one of many markers postulated for epithelial stem cells, p63, is challenged by widespread expression within stem cell-free regions, we examined p63 expression in these stem cell-associated cohorts compared with their controls. METHODS: Rabbit limbocorneal cryosections, cytospun cell-sorted (by fluorescence-activated cell sorter) side population (SP) and low side scatter (LSSC) cells, and limbal epithelial cells over feeders were stained for p63 by indirect immunofluorescence. Clones were fixed and stained daily for 7 days. Image analysis measured p63 intensity, plotting it against colony size. RESULTS: All basal limbal cells were positive for p63, yet only 5% to 7% expressed high p63 intensities, 40% intermediate, and the majority low. Side population cells were less than 1% of total cells. The average intensity of SP staining was three times that of controls. Subpopulations displaying stemlike features exhibited highest p63 expression. Replication rates of isolated cells differed. Day 5 colonies contained 256 (16 hours/cycle) to two (96 hours/cycle) cells. Whereas all cells were positive for p63, intensity in slow-cycling cells was three to four times that in rapidly proliferating congeners. Increased cell doublings did not decrease fluorescence. CONCLUSIONS: Results suggest that p63 concentration is maximal in stem cells and decreases with differentiation. High p63 levels seem to correlate with cells of the SP and LSSC phenotypes, indicating high cell stemness. With identification of stem cells, further studies can elucidate their use in supporting ocular surface health.

Asunto(s)