Bilateral diffuse lamellar keratitis following bilateral simultaneous versus sequential laser in situ keratomileusis.

AIM: To determine the difference in the incidence of bilateral diffuse lamellar keratitis (DLK) in patients undergoing simultaneous versus sequential laser in situ keratomileusis (LASIK) as an indication of intrinsic risk for inflammation. METHODS: A retrospective non-comparative case series of 1632 eyes that had undergone bilateral, simultaneous or sequential LASIK between April 1998 and February 2001 at a university based refractive centre by three surgeons. All cases that developed clinically evident DLK were identified and reviewed. In order to identify isolated cases and exclude those caused by environmental factors, when more than one patient in a given session developed DLK, the session was excluded. The main outcome measure was the incidence of unilateral and bilateral isolated, non-epidemic DLK. RESULTS: Of 1632 eyes, 126 eyes (7.7%) of 107 patients developed at least grade 1 DLK. In six operating sessions, DLK was observed in more than one patient per session, and on this basis 13 patients were excluded. 16 of the 94 remaining patients developed DLK in both eyes (17.0%). Six of 41 patients (14.6%) in the simultaneous group, versus 10 of 53 patients (18.9%) in the sequential group developed bilateral DLK (p >0.5). CONCLUSION: In isolated, non-epidemic bilateral DLK, a similar incidence was observed regardless of whether the surgery was simultaneous or sequential, suggesting an underlying intrinsic cause for DLK.

A phase III, placebo controlled clinical trial of 0.5% levofloxacin ophthalmic solution for the treatment of bacterial conjunctivitis.

AIM: To compare the efficacy and safety of levofloxacin 0.5% ophthalmic solution (Quixin) with placebo for treatment of bacterial conjunctivitis. METHODS: In this prospective, randomised, placebo controlled, double masked, multicentre study, 249 patients with bacterial conjunctivitis received either 0.5% levofloxacin (n = 126) or placebo (n = 123) for 5 days, administered every 2 hours on days 1-2, then every 4 hours on days 3-5. Cultures were obtained and signs/symptoms evaluated at baseline, interim, and final visits. The end point was the last evaluable observation. Primary microbial outcomes were based on culture results; clinical outcomes were based on resolution of cardinal signs. RESULTS: 117 patients (60 levofloxacin, 57 placebo) were evaluated. Microbial eradication rates were significantly greater with levofloxacin at all time points, reaching 90% at end point. In a subgroup analysis, differences in eradication rates at end point were most pronounced in children but were also statistically significant for levofloxacin in adults. Clinical cure rates were significantly greater with levofloxacin at final visit and end point. Statistically significant differences favouring levofloxacin were measured at end point for resolution of conjunctival discharge, bulbar conjunctival injection, palpebral conjunctival injection, burning/stinging, itching, and photophobia. Adverse events were similar between groups. Safety composite scores analysed by age indicated significantly fewer children on levofloxacin experienced worsening symptoms. CONCLUSIONS: Levofloxacin 0.5% ophthalmic solution is safe and effective for treatment of bacterial conjunctivitis.

Expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinase 1 and 2 in inflammation-induced corneal neovascularization.

PURPOSE: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMP-2, MMP-9 and TIMPs in the corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9 and TIMPs during the course of cauterization-induced corneal neovascularization in a rat model. METHODS: Neovascularization of rat corneas was induced by silver nitrate cauterization. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 was examined by immunohistochemistry and RT-PCR. The protein activities of MMPs and TIMPs were compared in pre- and postcauterization corneas by gelatin zymography and reverse zymography, respectively. RESULTS: MMP-2, TIMP-1 and TIMP-2 immunoreactivities were expressed in normal corneas, predominantly in the corneal epithelium. After injury, immunoreactivities of both MMPs and TIMPs were increased, notably in the healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts and ingrowing vascular endothelial cells. The increase in gross MMP-2 enzymatic activity paralleled the maximal vascular ingrowth on day 4, while the gross MMP-9 enzymatic activity rose immediately on day 1, then decreased steadily, which paralleled the magnitude of inflammatory cell infiltration. The immunoreactivity of MMPs/TIMPs decreased significantly 2 weeks after cauterization. On day 35, MMP-2, TIMP-1 and TIMP-2 staining was seen only in corneal epithelium and vascular endothelial cells. Both the RT-PCR and reverse zymography results revealed a more constant expression of TIMP-2, while the TIMP-1 expression appeared to be more inducible. CONCLUSION: MMPs as well as TIMPs were upregulated in cauterization-induced corneal neovascularization, suggesting that both may participate in extracellular matrix remodeling in the corneal wound healing, inflammation and neovascularization processes.

Objective assessment of transient corneal haze and its relation to visual performance after photorefractive keratectomy.

PURPOSE: Photorefractive keratectomy has the potential to cause transient corneal haze. The purpose of this study was to evaluate the relationship between transient corneal haze as measured by an objective means and high and low contrast visual performance. METHODS: In a prospective study, 44 eyes of 28 patients were examined preoperatively and at 1, 3, 6, and 12 months after photorefractive keratectomy. Five laser in situ keratomileusis and two intrastromal corneal ring segments (Intacs [KeraVision, Fremont, CA]) were included for comparison, because these procedures are not expected to cause haze. Haze was measured using a prototype objective hazemeter, TSPC-3, a modification of the Nidek EAS-1000. Visual performance was measured using high-contrast visual acuity and the Rabin Small Letter Contrast Test. RESULTS: Corneal haze was greatest at the 1-month examination and was consistent with a decrease in visual performance on both tests. Corneal haze resolved in 82% of eyes by 10 +/- 4 months after photorefractive keratectomy. However, visual performance had not returned to preoperative levels in 65% and 81% of these eyes on the high-contrast visual acuity test and the Small Letter Contrast Test, respectively. Eyes that underwent laser in situ keratomileusis and Intacs did not develop corneal haze; however, visual decrements were measured. CONCLUSIONS: As a clinical tool, the TSPC-3 hazemeter objectively measures very subtle changes in haze levels. Corneal haze appears to account for only approximately 50% of visual performance changes in the early healing period after photorefractive keratectomy. Other factors, namely topographic abnormalities, are more likely to be an important cause of persistent visual disturbances.

[Development of corneal storage medium--second report. Examination of human cornea].

PURPOSE: To develop and evaluate by histological methods a new corneal storage medium with a simple formula. METHODS: We compared a corneal storage medium which contained minimum essential medium and 2.5% chondroitin sulfate (molecular weight 27,500), pH 7.33, osmolality 320 mOsm/kg with OPTISOL-GS. Paired human donor eyes provided by the Lions Eye Bank of Oregon were stored in a moist chamber until the experiment. A cornea with scleral rim was excised and stored in OPTISOL-GS, and its fellow cornea was stored in the test medium for 5, 10, or 14 days at 4 degrees C. Histological examination of corneal endothelial cells was done by both scanning electron microscopy and transmission electron microscopy. RESULTS: On days 5 and 10, there was no significant difference in histological findings between corneas stored in OPTISOL-GS and those in the test medium. Both corneal groups developed degenerative changes with the increase of storage time, but their histological findings were similar for both storage media. On day 14, corneal endothelial cells showed marked degeneration of intracellular organelles such as a swelling of mitochondria in both media. CONCLUSION: Human corneas stored in the test medium for 14 days maintained their structure as well as those in OPTISOL-GS. This shows that the newly developed corneal storage medium composed can be used for medium-term corneal storage.

The intrastromal corneal ring segments: Intacs.

Although evolution of the intrastromal corneal ring technology will unquestionably continue, the ICRS in its current design is remarkably safe and offers several potential advantages, including preservation of the central cornea, reversibility and adjustability. The approval of this technology by the FDA has added a potentially unique procedure to the refractive surgeon's surgical inventory.

In vitro susceptibilities of bacterial ocular isolates to fluoroquinolones.

PURPOSE: To evaluate and compare the in vitro antimicrobial activity of levofloxacin versus ciprofloxacin and ofloxacin against ocular isolates from patients with bacterial conjunctivitis. METHODS: The in vitro antimicrobial susceptibilities of ocular isolates to levofloxacin, ofloxacin, and ciprofloxacin were determined using both the agar disk diffusion and broth dilution methods. RESULTS: Disk diffusion susceptibility testing disclosed that 99% (100 of 101 isolates) of gram-negative isolates and 98% (127 of 129 isolates) of gram-positive isolates were susceptible to levofloxacin; 96% (97 of 101 isolates) of gram-negative isolates and 78% (100 of 129 isolates) of gram-positive isolates were susceptible to ofloxacin; and 94% (95 of 101 isolates) of gram-negative isolates and 61% (79 of 129 isolates) of gram-positive isolates were susceptible to ciprofloxacin. Broth dilution testing disclosed that 99% (72 of 73 isolates) of gram-negative isolates and 98% (111 of 113 isolates) of gram-positive isolates were susceptible to levofloxacin; 96% (70 of 73 isolates) of gram-negative isolates and 92% (104 of 113 isolates) of gram-positive isolates were susceptible to ofloxacin; and 95% (69 of 73 isolates) of gram-negative isolates and 82% (93 of 113 isolates) of gram-positive isolates were susceptible to ciprofloxacin. CONCLUSIONS: In this study, levofloxacin demonstrated superior in vitro activity against human bacterial conjunctival isolates compared with either ofloxacin or ciprofloxacin (levofloxacin > ofloxacin > ciprofloxacin).

Flap haze after epithelial debridement and flap hydration for treatment of post-laser in situ keratomileusis striae.

PURPOSE: To report a case of corneal haze after attempts to eliminate post-laser in situ keratomileusis (LASIK) lamellar striae. METHODS: Case report. RESULTS: A 24-year-old woman with visually significant flap striae 2 months after LASIK underwent manual epithelial debridement and flap hydration, refloating, and stretching to eliminate the striae. Three weeks after this intervention, she developed visually significant haze that was confined to the stroma of the flap. The haze slowly improved with use of a topical steroid. CONCLUSION: Stromal haze can develop after treatment of flap striae with epithelial debridement and hypo-osmolar irrigation. We speculate that these maneuvers may have induced cell death of anterior keratocytes and led to haze formation, as can occur after simple epithelial debridement and epithelial scrape-photorefractive keratectomy.

Aspergillus fumigatus keratitis after laser in situ keratomileusis.

PURPOSE: To report a case of Aspergillus fumigatus keratitis after a laser in situ keratomileusis (LASIK) enhancement procedure. METHOD: Case report. RESULTS: A 56-year-old woman developed an ulcer in the flap 13 days after LASIK enhancement. A 4-week course of fortified antibiotics for a presumed bacterial infection followed. The ulcer progressed, causing 60% thinning of the corneal stroma. A biopsy was performed 5 weeks after onset of symptoms, and antifungal agents were initiated. Cultures showed A. fumigatus. Her cornea perforated after the biopsy, requiring cyanoacrylate and lamellar overlay sutures, but the infiltrate resolved on antifungal agents. CONCLUSION: This report is the first description of Aspergillus keratitis after LASIK. We hypothesize that the infection became established on the stromal bed during surgery and led to melting, anteriorly through the flap and posteriorly through the stroma. Diagnosis was made by a corneal biopsy and inoculation of a wide array of media. This case demonstrates the need to consider atypical organisms, including fungi, in the differential diagnosis of post-LASIK infections when there is no response to therapy and highlights the role of corneal biopsy and flap lifting in the diagnosis of this condition.

Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane.

PURPOSE: To identify the potential antiangiogenic and antiinflammatory proteins expressed in human amniotic membrane tissue. METHODS: Human amniotic epithelial and mesenchymal cells were isolated from human amniotic membranes by sequential trypsin and collagenase digestion. Total RNAs were harvested from freshly obtained human amniotic epithelial and mesenchymal cells. Antiangiogenic and antiinflammatory proteins were detected by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique and further confirmed by DNA sequencing of PCR-amplified transcripts. The distribution of tissue inhibitors of metalloproteinase (TIMPs) were studied further by immunohistochemistry performed on paraffin-embedded amniotic membrane tissue. RESULTS: RT-PCR results showed that both human amniotic epithelial and mesenchymal cells express interleukin-1 receptor antagonist, all four TIMPs, collagen XVIII, and interleukin-10. Thrombospondin-1 was expressed in all of the epithelial cell specimens and in one out of five mesenchymal cell specimens. Furthermore, immunohistochemistry studies performed on freshly prepared amniotic membrane confirmed that all members of the TIMP family were present in epithelial and mesenchymal cells as well as in the compact layer of the amniotic stroma. In cryopreserved amniotic membranes, positive staining was seen in residual amniotic cells and stroma. CONCLUSIONS: Human amniotic membrane epithelial and mesenchymal cells express various antiangiogenic and antiinflammatory proteins. Some of those proteins also were found in amniotic membrane stroma. These findings may explain in part the antiangiogenic and antiinflammatory effects of amniotic membrane transplantation.

Complex limbal choristomas in linear nevus sebaceous syndrome.

OBJECTIVE: This study aimed to describe the clinical and histopathologic findings in four patients with complex limbal choristomas associated with linear nevus sebaceous syndrome (LNSS), a rare disorder including nevus sebaceous, seizures, and mental retardation, and often accompanied by ocular anomalies. DESIGN: Small observational case series. METHODS: A retrospective review of the clinical and histopathologic records of four patients. RESULTS: Each of four patients had complex limbal choristomas in the setting of clinical and histopathologic LNSS. The limbal choristomas were multiple in three patients and bilateral in two patients. Most choristomas involved the superotemporal limbus (6 of 10), although nasal (3 of 10) and inferior (1 of 10) limbal tumors also were present. Three patients had significant corneal astigmatism or involvement of the central cornea requiring surgical removal of their choristomas, one accompanied by a lamellar keratoplasty and another accompanied by two consecutive penetrating keratoplasties. Each graft eventually vascularized, reducing vision. One patient's vision was limited by amblyopia and another by occipital cortical dysgenesis with visual impairment. Histopathologic examination of the excised choristomas showed foci of lacrimal gland (3 of 4 patients), adipose tissue (3 of 4), neural tissue (1 of 4), cartilage (1 of 4), lymphoid follicles (1 of 4), skin adnexal tissue (1 of 4), and smooth muscle (1 of 4). Other associated ocular findings included an eyelid mass (1 of 4), colobomas of the eyelid (3 of 4), colobomas of the choroid and retina (2 of 4), nonparalytic strabismus (2 of 4), scleral ectasia (1 of 4), partial oculomotor palsy with ptosis and anisocoria (1 of 4), microphthalmia (1 of 4), hypertelorism (1 of 4), and cortical visual impairment (1 of 4). CONCLUSIONS: Complex limbal choristomas, although rare, can occur in the setting of LNSS and can be associated with multiple ocular and systemic abnormalities. Visual prognosis appears poor in most cases despite aggressive management.

Core antigen mutations of human hepatitis B virus in hepatomas accumulate in MHC class II-restricted T cell epitopes.

Despite the extensive molecular information on serum-derived human hepatitis B viruses (HBV), liver-derived replicative HBV genomes have remained largely uninvestigated. We have examined the sequences of the entire core antigen (nucleocapsid) of liver-derived HBVs in 15 different hepatoma patients. Bona fide mutations, rather than subtype polymorphism, have been identified based on the high-frequency occurrence of structural differences from wild type at the highly evolutionarily conserved positions, instead of at the positions known to contain genetic heterogeneity among different isolates from different geographic locations. The distribution of these naturally occurring mutations of HBV core gene appears to be nonrandom and is found predominantly within three major (I, IV, and V) and four minor domains (II, III, VI, and VII). In general, domain IV mutations correlate with domain V mutations. The replicative HBV DNAs tend to accumulate a higher number of mutated core domains than the integrated HBV DNAs. At the domain level, there is no significant difference in HBV core mutation frequencies between the liver tumors and the adjacent nontumorous livers. Strikingly, domains I, III, and V coincide with three major known T cell epitopes within the core protein in acute and chronic hepatitis B patients. Furthermore, these domains coincide with HLA class II-restricted T cell epitopes, rather than with the conventional HLA class I-restricted epitopes of cytotoxic T lymphocytes. Our results support the hypothesis that HBV core antigen variants can accomplish immunoevasion via accumulated escape mutations. In addition, they also provide a potential molecular explanation for the maintenance of persistent infection of human hepatitis B virus in chronic carriers.

Suppression of interferon-gamma induction of MHC class II and ICAM-1 by a 26-base oligonucleotide composed of deoxyguanosine and deoxythymidine.

Interferon-gamma (IFN-gamma) is an important cytokine released by T lymphocytes and natural killer cells which is able to induce expression of class II MHC and ICAM-1, crucial factors in cellular immune response. HeLa S3, HS 27, and NF-71-1 are cell lines which can be induced to express HLA-DR and HLA-DP by exposure to IFN-gamma. When T2 (5'GGGGTTGGTTGTGTTGGGTGTTGTGTRNH(2)3') oligonucleotide was added at 5-20 microM every other day, cell surface induction of HLA-DR and HLA-DP by IFN-gamma was suppressed in a dose-dependent manner in HeLa S3. T2 suppressive effect on HLA class II was also observed in four different nontransformed human cell lines, HS 27 at passage 18, NF-71-1 at passage 5, human corneal endothelial cell at passage 5, and human retinal pigmented epithelial cell at passage 3. Control oligonucleotides had no suppressive effect. Northern hybridization showed that HLA-DR A mRNA induction by IFN-gamma was blocked by T2 in HeLa S3 and fibroblast 143B. The suppressive effect of T2 was also reversible as continued culture of the treated cells without further addition of the oligonucleotide allowed full re-expression of HLA-DR. Further experiments showed that T2 oligonucleotide was also able to inhibit IFN-gamma enhancement of ICAM-1 (CD54) on human corneal endothelial cell and human retinal pigmented epithelial cell. We conclude that T2 oligonucleotide is effective at suppressing HLA-DR, HLA-DP and ICAM-1 induction by IFN-gamma in transformed and nontransformed cells in vitro.

Detection of herpes simplex viral DNA in the iridocorneal endothelial syndrome.

OBJECTIVE: To test the hypothesis that the iridocorneal endothelial (ICE) syndrome has a viral origin by comparing the incidence of viral DNA in corneal specimens from patients with the ICE syndrome and from controls. DESIGN: Thirty-one corneas obtained from 25 patients with the ICE syndrome and six with chronic herpetic keratitis (n = 31) were compared with 30 control specimens obtained from 15 healthy donors and from 15 patients with other, nonviral chronic corneal diseases. METHODS: Primer pairs and polymerase chain reaction methods were used to identify and amplify either a segment of the DNA polymerase gene in the case of the herpes simplex and zoster viruses or a region of the nuclear antigen gene for the Epstein-Barr virus. The oligonucleotide amplified by polymerase chain reaction was fully characterized with the use of restriction enzyme, hybridization, and sequence analyses to determine that it contained the expected base pair sequence. RESULTS: Sixteen of 25 ICE syndrome specimens and four of six herpetic keratitis specimens were positive for herpes simplex virus (HSV) DNA. All nine ICE syndrome specimens tested were negative for the presence of DNA from the herpes zoster or the Epstein-Barr viruses. Controls were uniformly negative for HSV DNA whether they were obtained from ostensibly normal corneas (n = 15) or from corneas with intestinal keratitis, aphakic bullous keratopathy, or keratoconus (n = 15). Tissue samples cut from positive ICE syndrome specimens yielded negative results when retested after the endothelial layer was removed. These findings indicate that localization of HSV DNA is within the endothelium, the tissue primarily involved in the pathogenesis of the ICE syndrome. CONCLUSIONS: Polymerase chain reaction evidence shows that HSV DNA is present in a substantial percentage of ICE syndrome corneal specimens and that HSV-DNA is absent in normal corneas and in corneas from patients with three other chronic corneal diseases. These results provide direct evidence to support our hypothesis that the ICE syndrome has a viral origin. We discussed clinical implications, including possible therapeutic interventions.

Biological availability and nuclease resistance extend the in vitro activity of a phosphorothioate-3'hydroxypropylamine oligonucleotide.

Augmented biological activity in vitro has been demonstrated in oligonucleotides (oligos) modified to provide nuclease resistance, to enhance cellular uptake or to increase target affinity. How chemical modification affects the duration of effect of an oligo with potent activity has not been investigated directly. We postulated that modification with internucleotide phosphorothioates and 3' alkylamine provided additional nuclease protection which could significantly extend the biological activity of a 26 mer, (T2). We showed this analog, sT2a, could maximally inhibit interferon gamma-induced HLA-DR mRNA synthesis and surface expression in both HeLa and retinal pigmented epithelial cells and could continue to be effective, in the absence of oligo, 15 days following initial oligo treatment; an effect not observed with its 3'amine counterpart, T2a. In vitro stability studies confirmed that sT2a conferred the greatest stability to nucleases and that cellular accumulation of 32P-sT2a in both cell types was also greater than other T2 oligos. Using confocal microscopy, we revealed that the intracellular distribution of sT2a favored greater nuclear accumulation and release of oligo from cytoplasmic vesicles; a pattern not observed with T2a. These results suggest that phosphorothioate-3'amine modification could increase the duration of effect of T2 oligo by altering nuclease resistance as well as intracellular accumulation and distribution; factors known to affect biological availability.

Corneal allograft rejection after multiple blood transfusions.

We treated two patients with corneal allograft rejections, both of which occurred within a few weeks after administration of multiple blood transfusions. In the first case, a patient with a graft that had been previously clear for 11 years developed severe endothelial rejection six weeks after transfusion of ten units of packed red blood cells. In the second case, a graft that had been clear for 14 months irreversibly rejected two weeks after transfusion of six units of packed red blood cells. These cases suggest that multiple blood transfusions may be a risk factor for corneal allograft rejection.

Lamellar flap corneal biopsy.

A modified technique of corneal biopsy is described that allows a lesion in the deep corneal stroma to be accessed for scraping or biopsy while sparing the stromal tissue anterior to the lesion. This technique may be especially valuable in cases of deep corneal pathology in which traditional methods of corneal biopsy risk excessive removal of tissue, which, in turn, can lead to complications such as thinning or perforation.