Surfactant protein D in Pseudomonas aeruginosa keratitis

PURPOSE: To determine the importance of surfactant protein D in Pseudomonas keratitis. METHODS: The surfactant D status of wild-type and surfactant D-deficient Black Swiss mice was confirmed by PCR reactions and immunoblot assay. Mouse corneas were infected with one of three strains of P. aeruginosa. At 1, 2, 3, and 6 days postinfection, eyes were scored by slit-lamp examination and bacteria per cornea quantified. RESULTS: Infected wild-type mice had slit-lamp scores on 3 and 6 days postinfection that were significantly lower than those of surfactant D-deficient mice (p 相似文献   

Cathelicidin-deficient (Cnlp -/- ) mice show increased susceptibility to Pseudomonas aeruginosa keratitis

PURPOSE: To examine the clinical progression and innate immune responses during Pseudomonas aeruginosa (PA) keratitis in cathelicidin-deficient (KO) mice. METHODS: PA (ATCC 19660) keratitis was induced in KO mice and wild-type (WT) littermates generated on a 129/SVJ background. Clinical score and histopathology were used to monitor the progression of infection at postinfection (PI) days 1, 3, 7, 14, and 21. Mouse corneas were harvested for viable bacteria quantitation, and myeloperoxidase (MPO) assays were performed to determine the number of infiltrating neutrophils. ELISA was used to quantitate interleukin (IL)-1beta, IL-6, macrophage inflammatory peptide (MIP)-2, keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-alpha, and vascular endothelial growth factor (VEGF) levels in the corneas. RESULTS: WT mice were resistant (cornea healed), whereas KO mice showed increased susceptibility (corneas failed to recover by 21 days or perforated) to PA infection. Clinical scores were significantly elevated in the infected corneas of KO mice versus WT mice at 7, 14, and 21 days PI. Absence of cathelicidin resulted in significantly delayed clearance of PA in the cornea and an increased number of infiltrating neutrophils at 1, 3, 7, and 14 days PI. KO mice also exhibited differential expression of protein levels for IL-1beta, IL-6, MIP-2, KC, TNF-alpha, and VEGF up to day 21 PI compared with the WT mice. CONCLUSIONS: Cathelicidin-deficient mice showed considerable susceptibility to PA keratitis. The present study demonstrates direct in vivo evidence that endogenous expression of cathelicidin provides defense against corneal PA infection indicating its importance in host innate immunity at the ocular surface.     

Efficacy of topical gentamicin treatment after 193-nm photorefractive keratectomy in an experimental Pseudomonas keratitis model

Background: The treatment of Pseudomonas keratitis has many limitations, and further investigation to identify more effective approaches is required. We therefore studied the possible contribution of the debridement effect of 193-nm excimer laser on Pseudomonas keratitis in rabbit eyes. Methods: Pseudomonas keratitis was induced in 30 rabbit eyes by inflicting controlled central corneal scratches and applying a drop of Pseudomonas aeruginosa suspension. After 24 h, one cornea of each animal was photo-ablated (excimer laser: fluency 90 mJ/cm², 10 Hz, 213 pulses), yielding 50 m of tissue ablation, while the follow cornea served as control. Five groups of six animals each were formed and received: a subconjunctival injection of gentamicin 20, mg (group 1), topical 14 mg/ml gentamicin hourly (group 2) or every 2.5 h (group 3), or NaCl 0.9% hourly (group 4) for 8 h. In group 5, animals were sacrificed without additional treatment. After 9 h corneas were excised, homogenized, serially diluted, and plated on agar blood plates. The numbers of colony-forming units (CFU) per cornea were statistically evaluated (Mann-Whitney test). Results: In control eyes, a greater decrease of CFU was observed in group 2 than in group 3 (P = 0.03). In laser-ablated eyes, there was no difference in CFU between groups 2 and 3. Comparison of the excimer-treated and control eyes revealed a greater number of bacteria (CFU) in controls only in group 3 (P=0.02). Conclusion: Our study suggests that controlled debridement of cornea with excimer laser may improve the effect of topical antibiotics.Presented at ECORA First Annual Meeting, 5 October 1993, Bonn, Germany     

Recurrent herpetic keratitis in mice]

In order to investigate the mechanism of recurrence in herpes simplex keratitis, it is very important to establish an animal model. As a first step, mice were examined with the slit-lamp biomicroscope to determine whether they spontaneously showed recurrent epithelial keratitis after healing of primary herpetic keratitis. Among 90 eyes of 45 inbred C57BL/6 mice, recurrent epithelial keratitis stained with fluorescein was observed in 10 eyes of 9 mice during the observation period up to 50 days after the primary corneal infection with herpes simplex virus (HSV) type I Amakata strain (virulent strain). Recurrent epithelial keratitis was observed in 17 eyes of 15 mice among 116 eyes of 58 ddy mice infected with HSV-I Ska strain (avirulent strain). The epithelial lesions showed punctate or dendritic patterns and continued for one to 7 days. HSV antigen was detected by the fluorescent antibody technique in the cornea of 5 out of 8 eyes which showed recurrent epithelial keratitis using another ddy mice group tested. It was limited in the epithelium of the cornea. These results show that mice herpetic keratitis recurs spontaneously.     

Clarithromycin for experimental Staphylococcus aureus keratitis.

PURPOSE: Clarithromycin, a macrolide antibiotic not previously tested against the common causes of bacterial keratitis, was analyzed for its effectiveness in reducing the number of viable bacteria in a Staphylococcus keratitis model. An in vivo comparison of the effectiveness of clarithromycin to erythromycin, minocycline, and tetracycline for three strains of Staphylococcus aureus was done. METHODS: Rabbit eyes were intrastromally injected with 100 colony forming units of one of three strains of S. aureus. Two strains were methicillin-sensitive (ATCC 25923 and MSSA 309) and one strain methicillin-resistant (COL). Eyes were treated every 30 minutes with 0.3% clarithromycin, erythromycin, tetracycline, or minocycline from 4 to 9 hours postinfection. The number of colony forming units (CFU) per cornea in all eyes was determined at 10 hours postinfection. RESULTS: Vehicle-treated and untreated eyes (controls) contained over 6 logs of CFU per cornea, a value significantly higher than any of the antibiotic-treated eyes (P < or = 0.0001). Clarithromycin or erythromycin therapy significantly decreased the number of CFU per cornea by approximately 5 logs in the eyes infected with the methicillin-sensitive strains and by approximately 4 logs in the eyes infected with the methicillin-resistant strain. Tetracycline and minocycline were also successful in treating these strains, but overall showed less effectiveness than clarithromycin and erythromycin. CONCLUSIONS: Clarithromycin proved to be an effective ocular medication for the therapy of experimental S. aureus keratitis. The effectiveness of clarithromycin in this model and its known effectiveness for a variety of bacterial pathogens suggests a role for this drug as a useful ocular antibiotic.     

Lysostaphin treatment of methicillin-resistant Staphylococcus aureus keratitis in the rabbit

PURPOSE: To determine the efficacy of lysostaphin treatment of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a rabbit model. METHODS: The sensitivity to lysostaphin and vancomycin were compared for 34 MRSA and 12 methicillin-sensitive strains. Methicillin-resistant S. aureus strain 301 (MRSA 301) or a methicillin-sensitive strain of low virulence, ISP546, was intrastromally injected into rabbit corneas. Rabbit eyes were treated topically every 30 minutes from 4 to 9 or 10 to 15 hours postinfection with 0.28% lysostaphin or 5.0% vancomycin. Rabbits were killed and corneas were excised and cultured to determine the number of colony forming units (CFU) per cornea. RESULTS: Ninety percent minimal inhibitory concentrations were at least 19-fold lower for lysostaphin than for vancomycin. With early therapy (4 -9 hours postinfection) lysostaphin sterilized all MRSA 301-infected corneas, whereas untreated corneas contained 6.52 log CFU/cornea (P < or = 0.0001). Corneas infected with MRSA 301 and treated similarly with vancomycin retained 2.3 +/-0.85 log CFU/cornea, and none were sterile. When therapy was begun later (10-15 hours postinfection) the residual bacteria in lysostaphin-treated eyes were significantly less numerous than in vancomycin-treated eyes (0.58 +/- 0.34 vs. 5.83 +/- 0.16 log CFU/cornea, respectively; P < or = 0.0001). Three experiments were performed to demonstrate that lysostaphin penetrated the cornea to kill bacteria in vivo; lysostaphin-treated eyes were found to recover from infection, bacteria that did not cause epithelial defects (ISP546) were susceptible to lysostaphin, and inhibition of lysostaphin when harvesting corneas did not alter the observed therapeutic values of lysostaphin. CONCLUSIONS: Lysostaphin is very effective in treating keratitis mediated by methicillin-sensitive or methicillin-resistant S. aureus.     

In Vivo Imaging of Corneal Inflammation: New Tools for Clinical Practice and Research

Purpose: Infectious and inflammatory corneal diseases are a major cause of blindness. To date, assessment of corneal inflammation, has only been possible by slit-lamp biomicroscopy. The purpose of this study is to review the current state of imaging technologies enabling in vivo imaging of inflammation in the cornea.

Methods: Literature review of peer-reviewed articles on in vivo imaging modalities.

Results: Current means of diagnosis and treatment follow-up for immune and infectious keratitis are limited to slit-lamp biomicroscopy. Several modalities are currently emerging, allowing for in vivo imaging of corneal inflammation, including in vivo confocal microscopy, anterior segment optical coherence tomography, and intravital multiphoton microscopy.

Conclusion: Several in vivo imaging technologies are currently evolving, allowing for objective assessment of corneal inflammation and treatment response.     

Corneal virulence of Pseudomonas aeruginosa elastase B and alkaline protease produced by Pseudomonas putida

PURPOSE: To measure the specific virulence contributions of two Pseudomonas aeruginosa proteases, elastase B and alkaline protease, when expressed separately by Pseudomonas putida in a rabbit model of bacterial keratitis. METHODS: P. putida KT2440 was transformed with plasmids that enabled the extracellular production of either elastase or alkaline protease. Protease expression was confirmed by zymography and immunoblotting. P. putida expressing elastase, alkaline protease, or vector alone was injected intrastromally (10(3) colony forming units [CFU]) into rabbit corneas (n=6). Infected eyes were graded by slit-lamp examination (SLE) at 20, 24, 28, and 32 hr postinfection (PI). Rabbits were sacrificed at 33 hr PI, and the log CFU (+/-SEM) per cornea was determined. RESULTS: SLE scores for eyes infected with P. putida producing elastase were significantly higher than those infected with vector alone at all time points (por=0.1), but small erosions formed in 33% of corneas. At both 24 and 28 hr PI, the SLE scores for corneas infected with P. putida producing elastase were significantly higher than those infected with P. putida producing alkaline protease (p相似文献   

Age-related differences in rabbits during experimental Staphylococcus aureus keratitis

PURPOSE: To analyze age-related changes in susceptibility to experimental Staphylococcus aureus keratitis and purified alpha-toxin in rabbits. METHODS: Intrastromal injection of S. aureus (100 colony-forming units [CFUs]) induced keratitis in young (6-8 weeks) and aged (approximately 30 months) New Zealand White rabbits. Bacteria and polymorphonuclear leukocytes (PMNs) per cornea were quantified. Purified alpha-toxin at 1, 10, 25, or 50 hemolytic units (HU) or heat-inactivated alpha-toxin was intrastromally injected into corneas, and pathologic changes were determined by slit lamp examination (SLE) and histopathologic analysis. alpha-Toxin hemolysis assays were performed using erythrocytes from young and aged rabbits. RESULTS: S. aureus keratitis produced significantly higher SLE scores in young rabbits than in aged rabbits at 15, 20, and 25 hours postinfection (PI; P < or = 0.001); aged rabbits essentially recovered from S. aureus keratitis by 7 days PI. At 25 hours PI, numbers of CFUs and PMNs in corneas of young and aged rabbits were equivalent (P > or = 0.6); the bacterial burden in aged rabbits declined by 5 logs per cornea from day 1 to day 7 PI. Intrastromal injection of > or =10 HU alpha-toxin also produced significantly more disease in young than in aged rabbit corneas (P < or = 0.05), whereas 1 HU or heat-inactivated toxin yielded negligible pathologic changes in either group. Hemolysis assays of erythrocytes from young rabbits demonstrated greater susceptibility to alpha-toxin compared with those from aged rabbits. CONCLUSIONS: Corneas and erythrocytes of young rabbits, relative to aged rabbits, are significantly more susceptible to S. aureus keratitis and to alpha-toxin.     

Herpetic keratitis in inbred mice

The corneas of four inbred strains of mice (BALB/c, DBA/2, C3H and C57BL/6) were inoculated with the RE strain of herpes simplex virus, type 1. The corneas were examined at frequent intervals and graded on a scale of 0 (clear cornea) to +5 (severe necrotizing stromal keratitis). At 3 weeks postinfection, the mean corneal scores were: DBA/2, 4.0; BALB/c, 2.2; C3H, 0.7; and C57BL/6, 0.15. The differences between the scores are statistically significant (P less than 0.05), except for the C3H and C57BL/6 strains. The order of severity of corneal disease in these mice corresponds to the order of susceptibility to systemic infection found in these same inbred strains. Additional studies of herpetic keratitis in inbred mice should prove helpful in understanding the genetic and immunologic basis of herpetic stromal keratitis.     

Methicillin-resistant Staphylococcus aureus keratitis in the rabbit: therapy with ciprofloxacin, vancomycin and cefazolin.

To determine the efficacy of a fluoroquinolone antibiotic in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) keratitis, topical administration of 0.3% ciprofloxacin was compared with topical 5.0% vancomycin or 5.0% cefazolin in experimental infections in the rabbit eye. The infections were established by intrastromal injection of 100 colony forming units (CFU) of MRSA, which resulted in greater than 10(6) CFU per cornea by 12 hr postinfection. Chemotherapy (one drop every 15 min) was given from 4-9, 10-15, or 10-20 hr postinfection. Early therapy (4-9 hr postinfection) with ciprofloxacin rendered all eyes free of bacteria; ciprofloxacin was significantly more effective than vancomycin or cefazolin. When treatment was initiated 6 hr later (10-15 hr postinfection), no corneas became free of bacteria, but ciprofloxacin was again more effective than vancomycin or cefazolin. Bacterial killing by ciprofloxacin after treatment from 10-20 hr postinfection was also significantly greater than that of vancomycin. Overall, the results show that ciprofloxacin is effective in killing methicillin-resistant staphylococcus aureus, and is most effective when applied during the very early stages of infection.     

Increased severity of Pseudomonas aeruginosa corneal infection in strains of mice designated as Th1 versus Th2 responsive

PURPOSE: Mice favoring Th1 (C57BL/6, C57BL/10, and B10.D2/nSn) versus Th2 (BALB/c, BALB/cBy, BALB.B, and BALB.K) response development were evaluated for their response to infection with Pseudomonas aeruginosa. This study addresses the question of whether Th1 versus Th2 response propensity affects the pathogenesis of bacterial keratitis in mice. METHODS: Ocular disease was determined by mean clinical score, slit lamp, plate counts, and histopathology, and antigen-specific cellular responses were assessed by immunostaining and measurement of delayed type hypersensitivity (DTH). RESULTS: Strains of mice favoring Th1 (B6, BL10, and B10.D2) versus Th2 (BALB/c, BALB/cBy, BALB.B, and BALB.K) responsiveness were infected with P. aeruginosa. Mice favoring Th1 response development exhibited a similar course of disease and the infected eyes of all mice perforated by 7 days postinfection (p.i.). Strains (BALB/c, BALB/cBy, BALB.B, and BALB.K) favoring Th2 response development exhibited a milder course of disease, and none of the infected corneas perforated at 7 days p.i. In a Th1-responsive strain (B10.D2), positive immunostaining for CD4+ and CD8+ T cells was observed in the cornea by 3 days p.i. and by 5 days p.i., respectively, some cells stained positively for IL2-R, indicating that the cells were activated. In contrast, in a Th2 responder strain (BALB/c), there was no detectable positive immunostaining in cornea for any of the T-cell markers tested and DTH was significantly elevated in B10.D2 versus BALB/c mice. CONCLUSIONS: These studies are the first to provide evidence that in P. aeruginosa ocular infection, mouse strains favoring development of a Th1-type response are susceptible (cornea perforates), whereas strains favoring Th2 response development are resistant (no corneal perforation).     

A controlled study of amniotic membrane transplantation for acute Pseudomonas keratitis

ObjectiveTo evaluate the efficacy of amniotic membrane transplantation (AMT) to improve the outcomes of acute Pseudomonas keratitis as compared with a control group.DesignProspective interventional case series with retrospective controls.ParticipantsWe studied 14 eyes with Pseudomonas keratitis as the AMT group and 11 eyes with Pseudomonas keratitis as the control group.MethodsEyes in the AMT group were treated with antibiotic therapy followed by single-layer AMT at 2 to 3 days. Eyes in the control group received only antibiotic therapy. Patients were followed for 11.1 ± 2.4 months.ResultsIn the AMT group, pain significantly decreased from a mean score of 2.4 ± 0.5 preoperatively to 1.1 ± 0.9 at day 2 postoperatively (p < 0.001). Corneal epithelial defects healed completely within 13.2 ± 2.6 days in the AMT group compared with 15.5 ± 3.4 days in the control group (p = 0.07). At final follow-up visits, the sizes of corneal opacity and deep neovascularization were not different between the 2 groups. However, the mean score for density of the corneal opacity was significantly less in the AMT group compared with the control group (2.1 ± 0.4 vs 2.5 ± 0.7, respectively, p = 0.04). Although the best corrected visual acuity using hard contact lenses was not different between the 2 groups, uncorrected visual acuity was better in the AMT group (0.45 ± 0.22 logMAR) than in the control group (0.71 ± 0.32 logMAR, p = 0.03). No patient in either group developed significant corneal thinning or perforation.ConclusionsAMT in acute Pseudomonas keratitis was associated with immediate pain relief, less density of the final corneal opacity, and better uncorrected visual acuity at the final follow-up visit.     

The Role of Nitric Oxide in Resistance to P. aeruginosa Ocular Infection

Purpose: This study determined the role of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in the resistance response of BALB/c mice to P. aeruginosa-induced keratitis. Methods: RT-PCR, nitrite detection, iNOS inhibition, ELISA, and immunohistochemistry were used. Results: Early after infection, iNOS mRNA expression and nitrite levels in cornea were elevated compared to levels in the uninfected cornea. Treatment with aminoguanidine sulfate (AG), an inhibitor of iNOS, resulted in extensive corneal destruction, reduced nitrite levels, and reduced nitrotyrosine staining. Infected mice also had increased bacterial burden and elevated levels of MIP-1α, IL-1β, and MIP-2 in the cornea. Dual-labeling immunohistochemistry established the macrophage as the major source of iNOS in the infected cornea. Conclusions: These data provide evidence that iNOS is constitutively expressed in the BALB/c cornea; that iNOS-derived NO is required for bacterial killing/stasis; and that the macrophage is the major cell source of NO.     

Susceptibility of aged mice to Staphylococcus aureus keratitis

PURPOSE: To determine the effect of age on the extent of pathogenesis of Staphylococcus keratitis in the mouse. METHODS: Corneas of young and aged mice (BALB/c, A/J, and C57BL/6) were scarified and topically inoculated with S. aureus. Slit lamp examination (SLE) and histopathology were performed, and bacterial colony forming units and myeloperoxidase activity were determined. RESULTS: SLE scores of infected eyes of aged mice were significantly higher at days 1 and 3 postinfection (PI) as compared to infected young mice. Histopathological changes observed in all aged mice were more severe than those in young mice. Young BALB/c and A/J mice demonstrated minimal signs of keratitis by day 3 PI, whereas aged mice of both strains demonstrated severe keratitis by day 3. Young C57BL/6 mice showed no clinical signs of keratitis, whereas aged C57BL/6 mice demonstrated moderate keratitis. CONCLUSIONS: Aged mice with S. aureus keratitis demonstrated increased pathology as compared to young mice.     

Effectiveness of topical taurolidine versus ciprofloxacin, ofloxacin, and fortified cefazolin in a rabbit Staphylococcus aureus keratitis model

PURPOSE: Taurolidine is a broad-spectrum, non antibiotic antimicrobial agent, not previously tested against the common causes of bacterial keratitis. This study, employing an experimental rabbit model of Staphylococcus aureus keratitis, investigated the effectiveness of topical taurolidine in reducing the number of bacteria, and its effectiveness was compared with topical ciprofloxacin, ofloxacin, and 5% cefazolin. METHODS: The right corneas of all rabbits were intrastromally injected with 100 colony-forming units of Staphylococcus aureus ATCC strain 25923. The animals were divided into the following seven groups: Group 1 (6 rabbits) received taurolidine, group 2 (6 rabbits) received ciprofloxacin, group 3 (6 rabbits) received ofloxacin, group 4 (6 rabbits)received cefazolin, group 5 (5 rabbits) received polyvinylpyrrolidone (vehicle),group 6 (4 rabbits) received sterile water, and group 7 (4 rabbits) was left un-treated (control group). The eyes were topically treated every 30 min with the above-mentioned substances from 4 to 9 h postinjection. One hour after the last drop administration (at 10 h postinjection), signs of inflammation were scored in a masked fashion by slit-lamp examination. Then, their corneas were processed. The number of colony-forming units (cfu) per cornea in all eyes was also determined. RESULTS: All antimicrobial (taurolidine, ciprofloxacin, ofloxacin, and cefazolin) treatments significantly reduced cfu numbers and slit-lamp examination scores compared with untreated eyes, eyes that received the vehicle, or eyes with sterile water (all p values <0.05). Regarding cfu numbers, although taurolidine therapy was significantly less effective than ciprofloxacin or ofloxacin,there was no significant difference between taurolidine and cefazolin groups.However, taurolidine had similar clinical examination scores with the other antimicrobials, while it had lower scores than the vehicle, sterile water, or un-treated eyes. CONCLUSIONS: The results obtained in this study suggest that topicaltaurolidine is an effective, novel ocular chemotherapeutic agent for the therapy of rabbit experimental Staphylococcus aureus keratitis. This drug may be a useful and promising ocular antimicrobial.     

Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa–Induced Keratitis

PurposePreviously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential.MethodsEight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid–modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-β-III tubulin antibody.ResultsAnti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1β at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII⁺ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression.ConclusionsProphylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.     

Effect of topical povidone-iodine versus topical ofloxacin on experimental Staphylococcus keratitis

Purpose: To compare the antibacterial effect of povidone-iodine (PI) with that of ofloxacin in an experimental model of bacterial keratitis.Methods: Staphyloccocal keratitis was induced in 21 eyes of Dutch Belted rabbits by intrastromal inoculation of approximately 280 organisms of Staphylococcus aureus. Six hours later, the animals were divided in four groups treated topically with saline 0.9%, Betadine 10%, Betadine 0.5% or Ofloxacin 0.3% (2 gtt every 30 min for 8 h). The central 8-mm cornea was excised, washed and homogenized. Colony counts were performed on serial 10-fold dilutions plated on blood and brain infusion agar and incubated overnight.Results: Colony-forming units per cornea were 7.4×10⁷ for the saline group compared to 8.2×10⁷ for PI 10% (P>0.5), 4.3×10⁷ for PI 0.5% (P<0.01) and no organisms for ofloxacin 0.3%.Conclusions: Betadine 0.5% demonstrates a statistically significant bactericidal effect compared with untreated staphyloccocal keratitis in our experimental model. Ofloxacin has superior antibacterial effect under the conditions studied. Further improvements in the povidone-iodine formulation are warranted prior to consideration for human keratitis. Received: 1 July 1999 Revised: 9 November 1999 Accepted: 10 November 1999     

Impaired eosinophil recruitment to the cornea in P-selectin-deficient mice in Onchocerca volvulus keratitis (River blindness)

PURPOSE: A murine model of helminth-induced keratitis (river blindness) that is characterized by a biphasic recruitment of neutrophils (days 1-3) and eosinophils (days 3+) to the cornea has been developed. The purpose of this study was to determine the relative contribution of P- and E-selectin in recruitment of these inflammatory cells from limbal vessels to the corneal stroma. METHODS: P- and E-selectin gene knockout (-/-) mice were immunized with antigens extracted from the parasitic helminth Onchocerca volvulus. One week after the last immunization, parasite antigens were injected directly into the corneal stroma. Mice were killed on days 1 and 3 postchallenge, and eyes were immunostained with either anti-eosinophil major basic protein (MBP) or with anti-neutrophil Ab. The number of cells in the cornea was determined by direct counting. RESULTS: Recruitment of eosinophils to the cornea was significantly impaired in P-selectin(-/-) mice (63.9% fewer eosinophils on day 1 [P: = 0.0015], and 61% fewer on day 3 [P: < 0.0001]) compared with control C57BL/6 mice. In contrast, P-selectin deficiency had no effect on neutrophil recruitment to the cornea. There was no inhibition of eosinophil and neutrophil migration to the corneas of E-selectin(-/-) mice, indicating that there is no direct role for this adhesion molecule in helminth-induced keratitis. CONCLUSIONS: The present study demonstrates that P-selectin is an important mediator of eosinophil recruitment to the cornea. P-selectin interactions may therefore be potential targets for immunotherapy in eosinophil-mediated ocular inflammation.