Epithelial proliferative potential of organ cultured corneoscleral rims; implications for allo-limbal transplantation and eye banking.

AIMS: To determine the epithelial proliferative capacity of organ cultured limbal tissue and correlate this with various donor and eye banking factors. METHODS: 24 corneoscleral limbal (CSL) rims left over from penetrating keratoplasty were split in half and set up as in vitro explant cultures. Corneal epithelial proliferative potential (CEPP) was assessed by the number of "cycles" of growth achieved before explants underwent exhaustion and failure to generate an epithelium to subconfluence. The dependence of CEPP on the age of the donor, time of death to enucleation, time of enucleation to organ culture, and time in organ culture in the eye bank was determined. RESULTS: CSL rims were capable of up to four cycles of culture with a wide variation between tissue samples. Of the various factors examined, death to enucleation time was the only statistically significant factor affecting the CEPP (regression coefficient: -0.062 (cycles/hour), CI -0.119 to -0.004, p = 0.037). Time in organ culture had little effect on CEPP. CONCLUSIONS: Preselected organ cultured CSL rims from eye banks may offer a viable alternative tissue source for use in allo-limbal transplantation.

Incidence of keratitis of varying severity among contact lens wearers.

AIM: To determine the incidence of non-severe keratitis (NSK) and severe keratitis (SK) among wearers of current generation contact lenses. METHODS: A 12 month, prospective, hospital based epidemiological study was conducted by examining all contact lens wearers presenting with a corneal infiltrate/ulcer to a hospital centre in Manchester. A clinical severity matrix was used to differentiate between NSK and SK, based on the severity of signs and symptoms. The size of the hospital catchment population and the wearing modalities (daily wear (DW) or extended wear (EW)) and lens types being used were estimated from relevant demographic and market data. RESULTS: During the survey period, 80 and 38 patients presented with NSK and SK, respectively. The annual incidences (cases per 10,000 wearers) for each wearing modality and lens type were: DW rigid--NSK 5.7, SK 2.9; DW hydrogel daily disposable--NSK 9.1, SK 4.9; DW hydrogel (excluding daily disposable)--NSK 14.1, SK 6.4; DW silicone hydrogel--NSK 55.9, SK 0.0; EW rigid--NSK 0.0, SK 0.0; EW hydrogel--NSK 48.2, SK 96.4; EW silicone hydrogel--NSK 98.8, SK 19.8. The difference in SK between EW hydrogel and EW silicone hydrogel was significant (p = 0.04). CONCLUSIONS: A clinical severity matrix has considerable utility in assessing contact lens related keratitis. There is a significantly higher incidence of SK in wearers who sleep in contact lenses compared with those who only use lenses during the waking hours. Those who choose to sleep in lenses should be advised to wear silicone hydrogel lenses, which carry a five times decreased risk of SK for extended wear compared with hydrogel lenses.

Abnormal prion protein in the retina of the most commonly occurring subtype of sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Involvement of the eye has been reported in patients with variant Creutzfeldt-Jakob disease (vCJD), but there is disagreement on whether retinal involvement occurs in sporadic Creutzfeldt-Jakob disease (sCJD). METHODS: Western blotting, paraffin embedded tissue blotting, and immunohistochemistry were used to test whether the abnormal form of the prion protein (PrPSc) accumulates to detectable levels in the eye in a case of the most common subtype of sCJD (MM1). RESULTS: Low levels of PrPSc were detectable in the retina, localised to the plexiform layers of the central retina. PrPSc was not detectable in other ocular tissues. CONCLUSIONS: The abnormal form of the prion protein is present in the retina in the most common sCJD subtype (MM1), albeit at levels lower than those found previously in vCJD and in sCJD of the VV2 subtype.

Apolipoprotein E and herpes virus diseases: herpes simplex keratitis.

Our previous studies showed that herpes simplex type 1 virus (HSV1) is present in a high proportion of the brain of elderly normal people and Alzheimer's disease (AD) patients. We subsequently discovered that the combination of HSV1 in brain and carriage of the type 4 allele of the gene for apolipoprotein E (apoE-epsilon 4) is a strong risk factor for AD, and also that apoE-epsilon 4 is a strong risk factor for herpes labialis. In this study we have examined apoE genotypes of sufferers from another disorder caused by HSV1, namely, herpes simplex keratitis (HSK), to find if an apoE allele is involved in the disorder. In 46 HSK patients the apoE-epsilon 4 allele frequency was 15%-the same as that found in 238 unaffected controls. The apoE-epsilon 2 allele frequency was 13%-higher than the value of 7% for unaffected people, but the difference is not statistically significant.

Fas mRNA expression in blood is reduced during episodes of human corneal graft rejection.

BACKGROUND: Our purpose is to examine levels of Fas mRNA expression in blood during human corneal transplant rejection. METHODS: Fas mRNA expression was detected by reverse transcription-PCR in blood from normal controls, corneal recipients at the time of transplantation and during episodes of rejection. RESULTS: Samples taken at the time of a corneal rejection episode showed Fas mRNA levels were significantly lower in these patients than either normal controls (P = 0.017) or corneal transplant recipients not undergoing graft rejection (P = 0.00052). Serial samples from five patients who suffered an episode of rejection showed that the level of Fas mRNA is reduced during the rejection episode and subsequently recovers. CONCLUSIONS: These results indicate low levels of Fas mRNA in blood may have a role in corneal transplant rejection.

Apoptosis in the endothelium of human corneas for transplantation.

PURPOSE: To determine whether endothelial cell loss of human corneas stored in organ culture before transplantation is due to apoptosis. METHODS: The corneal endothelium of human corneas, stored in organ culture at 34 degrees C for varying periods of time, were analyzed for the presence of apoptotic cells using the TdT-mediated dUTP nick-end labeling (TUNEL) technique. Corneal endothelial cell apoptosis was confirmed by Hoechst staining and immunolabeling with anti-caspase 3 active antibody. RESULTS: Apoptotic cells were identified in the corneal endothelium of human organ cultured corneas: their number and distribution demonstrated a close correlation with corneal folding and overall quality of the corneal endothelium. TUNEL-positive labeling of cells was confirmed as apoptotic by the presence of morphologic nuclear alterations identified by Hoechst staining and the presence of immunostaining for caspase-3 activity. Corneal endothelial cell apoptosis was independent of cause of donor death, death to enucleation time, and death to culture times. CONCLUSIONS: Corneal endothelial cell apoptosis appears to determine the suitability of a cornea for transplantation.

Multiplex polymerase chain reaction for diagnosis of viral and chlamydial keratoconjunctivitis.

PURPOSE: To develop a multiplex polymerase chain reaction (PCR) for the detection of adenovirus, herpes simplex virus, and Chlamydia trachomatis in conjunctival swabs. METHODS: Oligonucleotide primers for detection of the 3 agents were combined in one reaction and evaluated for optimal performance using control DNAs of adenovirus type 2, herpes simplex virus, and C. trachomatis plasmid. The multiplex PCR was evaluated prospectively against its corresponding uniplex PCRs, virus isolation, Chlamydia Amplicor PCR, and an immunoassay technique (immune dot blot test) in a total of 805 conjunctival swabs from patients with suspected viral and chlamydial keratoconjunctivitis. RESULTS: The multiplex PCR was as sensitive as uniplex PCRs for the detection of the agents in clinical specimens. In the prospective study, 48 of 49 (98%) clinical specimens were positive for adenovirus by the multiplex PCR compared with 26 of 49 (53%) by adenovirus isolation. For herpes simplex virus detection, the multiplex PCR had a sensitivity of 92% (34/37) compared with 94.5% (35/37) by cell culture. The multiplex PCR produced identical results to the Amplicor PCR (21/21; 100%) compared with 71% (15/21) by the immune dot blot test. CONCLUSIONS: With clinical specimens the multiplex PCR was as sensitive as its respective uniplex PCRs but more sensitive than adenovirus isolation and as sensitive as herpes simplex virus isolation or C. trachomatis Amplicor PCR. It has the potential to replace several diagnostic tests with consequent savings in cost. The test also reduces the risk of misdiagnosis by the clinicians.

Corneal sensitivity and substance P in experimental herpes simplex keratitis in mice.

Experimental herpes simplex keratitis in the mouse produced a rapid fall in both corneal sensitivity and levels of corneal substance P (SP). This finding supports the association of SP with sensory neurones and shows that such levels can be used as an indication of damage to neurones resulting, for example, from infection with HSV. However, the delay in recovery of SP compared to the more rapid and complete recovery of sensitivity suggests that SP in the cornea is not directly involved in mediation of the blink reflex.

Corneal epithelial-specific cytokeratin 3 is an autoantigen in Wegener's granulomatosis-associated peripheral ulcerative keratitis.

PURPOSE: In a previous investigation it was demonstrated that circulating antibodies to a 66-kDa corneal epithelial antigen (BCEA-A) are associated with peripheral ulcerative keratitis (PUK) in patients with Wegener's granulomatosis (WG). The aim of this study was to identify BCEA-A. METHODS: The 66-kDa antigen was purified from a bovine corneal epithelial protein extract, using DE52 ion exchange chromatography. Purified protein was used to raise rabbit polyclonal antibodies. These antibodies were used to screen a bovine corneal epithelial cDNA expression library. Positive clones were purified and sequenced. Clones were identified by DNA sequence homology searches of the GenBank DNA database. RESULTS: A cDNA clone that demonstrated strong binding to both the rabbit polyclonal antibody and patient sera, showed 85% homology to rabbit cytokeratin 3 (K3). K3 is a basic cytokeratin specific to corneal epithelium. No bovine DNA sequence for K3 is available. However, bovine K3 is larger than rabbit K3, with a molecular weight of 66 kDa. Immunofluorescence using both patient sera and the rabbit antibody demonstrated a cytoplasmic binding pattern on human corneal epithelium. CONCLUSIONS: This evidence suggests that the 66-kDa autoantigen (BCEA-A) associated with PUK in WG is cytokeratin 3, and this may form the basis of a diagnostic/prognostic test.

Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.

PURPOSE: To evaluate newly designed primers in a polymerase chain reaction (PCR) for the detection of adenovirus DNA in conjunctival swabs. METHODS: Oligonucleotides were derived from the adenovirus hexon gene and modified such that a maximum of only two mismatches occurred with adenovirus types 2 through 5, 7, and 16. Specificity was determined against adenovirus types 2 through 4, 7, 8 through 11, 14, 19, 37, 40, and 41 and from non-adenoviral DNA and the sensitivity by PCR amplification of purified adenovirus type 2 DNA. The assay was compared retrospectively with cell culture and a PCR with different primers on 59 stored conjunctival swab samples. The new PCR also was used prospectively in comparison with cell culture on 2743 conjunctival swabs. RESULTS: The 140-bp product was amplified from all the adenovirus serotypes tested except types 40 and 41, which have not been isolated from the eye. There were no amplified products from the non-adenoviral DNA tested. With adenovirus type 2 DNA, despite two deliberate mismatches, 40 copies of the target were detectable after PCR and ethidium bromide-staining. In the retrospective study, 51 of 55 (92.7%) were positive by this new PCR compared with 42 of 55 (76.4%) by the older PCR and 40 of 55 (72.7%) by cell culture. In the prospective study, the new PCR detected 386 of 415 (93%) adenovirus-positive specimens compared with 248 of 415 (59.8%) by cell culture. Of 167 specimens positive for herpes simplex virus by cell culture, none were positive by the adenovirus PCR. CONCLUSIONS: PCR with the newly designed primers shows a much increased sensitivity over cell culture and previous PCRs for the detection of adenoviruses in conjunctival swabs.

Characterization of two corneal epithelium-derived antigens associated with vasculitis.

PURPOSE: In a previous investigation into corneal autoimmunity, it was demonstrated that a putative autoantigen, a protein of 66 kDa, present in bovine corneal epithelium, binds circulating autoantibodies in approximately 60% of patients with Wegener's granulomatosis (WG). The aim of the present study was to characterize and identify the 66-kDa protein. METHODS: A purification protocol was established for the 66-kDa protein using standard chromatography techniques. During the purification procedure it became clear that the 66-kDa protein detected in patients' sera was in fact two proteins, both running at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that eluted in different fractions on DE-52 chromatography columns. These two proteins have been labeled bovine corneal epithelial antigen-A and -B (BCEA-A and BCEA-B). Further investigations of antibody binding have demonstrated that patients' sera bind to either one or the other of these proteins with no cross-reactivity between them. Separated BCEA-A and BCEA-B protein extracts were immunoblotted with 27 WG patients' sera, 10 Churg-Strauss syndrome (CSS) patients' sera, 31 rheumatoid arthritis (RA) patients' sera, and 40 healthy control subjects' sera from the blood bank. RESULTS: Forty-six percent of WG patients' sera had antibodies to one of the 66-kDa antigens, whereas none of the healthy control subjects' sera had 66-kDa antibodies (P < 10(-5)). In the WG group, 31% were positive to BCEA-A (versus controls, P = 0.0023), and 15% were positive to BCEA-B. WG patients with peripheral ulcerative keratitis (PUK) had a significant association with anti-BCEA-A antibodies when compared with healthy control subjects (50%, P < 10(-6)). However, in the RA group with no eye disease there was an association with BCEA-A (25%, P = 0.011) but not in the RA group with PUK. The frequency of anti-BCEA-B antibodies was significantly increased in patients with CSS (60%, P < 10(-7)). CONCLUSIONS: In summary, it has been shown that vasculitis patients have antibodies to two 66-kDa corneal antigens and that autoantibodies to these antigens are mutually exclusive. It has also been shown that antibodies to BCEA-B are associated with CSS, whereas BCEA-A antibodies are associated with WG and RA.

Ocular infection with herpes simplex virus in nonimmune and immune mice.

In a detailed study of ocular infection by herpes simplex virus (HSV) type 1 in mice, the course and signs of eye disease were investigated and compared in primary and secondary infection using slit-lamp examination, culture of the tear film, and monitoring of the blink reflex. Response to primary inoculation ranged from subclinical infection to severe keratitis. Compared with conjunctival scarification, corneal scarification resulted in more frequent and severe eye disease and signs of CNS infection. Previous infection in the skin of the contralateral ear considerably modified subsequent infection of the eye so that signs of disease occurred earlier, were limited to dendritic keratitis with some stromal involvement, and were largely reversible. The mouse seems to be a suitable animal for studying ocular infection with HSV.

Clinical, biochemical, and immunohistochemical features of necrobiotic xanthogranulomatosis.

AIMS: To describe the clinical features of two patients with paraproteinaemia and necrobiotic xanthogranulomatosis together with detailed immunohistochemistry of the lesions in one. METHODS: The clinical history and results of biochemical investigations of the patients were retrieved from the files. Immunohistochemistry was used to investigate the expression of macrophage and mast cell markers, amyloid A and P, S-100 protein, and apolipoprotein AI and B in xanthogranulomatous skin lesions from patient 2. In addition, protein A-sepharose chromatography was used to separate serum from patient 2 and apolipoprotein B and the IgG paraprotein were measured in the fractions eluted. RESULTS: Monocytes/macrophages comprised the major cellular component of the lesion, and unusually for xanthomata, areas of collagen necrosis were also seen. Activated mast cells were present at the margins of macrophage clusters and adjacent to areas of collagen necrosis. Serum paraprotein was bound to low density lipoproteins as judged by protein A-sepharose chromatography, and was also located within macrophagic foam cells of the lesion on immunohistochemistry. CONCLUSIONS: These observations demonstrate many features similar to atherosclerosis including collagen necrosis and mast cell activation.

Eye care for the critically ill.

PURPOSE: To evaluate the effectiveness and efficiency of an algorithm in the prevention of ocular surface disease in sedated and unconscious patients in the intensive care unit (ICU). METHODS: The eyes of all sedated and unconscious patients admitted to an ICU between September and December 1998 were managed according to an eye care algorithm. The applications of the preventive measures were assessed by a single observer twice weekly. The lid position, the presence and degree of keratopathy, sedation score, and the treatment received were documented at every assessment. RESULTS: Thirty-four patients were recruited for management according to the eye care algorithm over a period ranging from 1 to 28 days. Four patients were excluded because of failure by staff to adhere to the protocol. In 18 patients no active treatment was required. Out of six patients who had conjunctival exposure and were given ocular lubricants, four maintained clear corneas. No corneal or conjunctival staining was noted in two of the four patients whose lids required closure with Micropore tape for corneal exposure. Use of lid taping and lubricants prevented corneal abrasion in two patients who were prone ventilated. The prevalence of ocular surface abnormalities was 8.7 % where the algorithm was properly followed (23 patients). CONCLUSION: The proposed eye care algorithm appears to be effective in preventing ocular surface abnormalities in the sedated and unconscious patients in the ICU, and efficient in that it may reduce the workload required for critically ill patients.

Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs.

Adenoviruses and herpes simplex virus (HSV) can cause clinically indistinguishable episodes of acute eye disease. Adenovirus infection is associated with nosocomial outbreaks and HSV may result in episodes of recurrent ocular inflammation. In a comparison of multiplex PCR for the two viral DNAs and virus isolation in cell culture, identical results were obtained for 18 of 20 specimens (positive for adenovirus in 5, HSV in 5, and negative in 8). One specimen was falsely negative for each viral DNA. Inclusion of human beta-globin primers in the adenovirus-HSV reaction was precluded by a consequential 10--100-fold reduction in sensitivity for the two viral targets and by the failure of beta-globin DNA amplification at the annealing temperature (45 degrees C) required to ensure detection of adenoviruses of serotypes 7 and 11 with the selected adenovirus primers. A single-target beta-globin PCR gave positive results with 19 of the 20 specimens prepared by treatment with proteinase K lysis buffer, indicating the effectiveness of this simple DNA extraction procedure. Nonetheless, the availability of effective antiviral therapy for HSV made monitoring for extraction failure using human primers crucial to avoid false-negative results for HSV DNA. Adenovirus-HSV PCR has considerable potential for the rapid diagnosis of viral eye disease particularly if beta-globin primers can be included in the reaction.

Substance P in the mouse cornea: effects of chemical and surgical denervation.

The substance P content of the cornea of the adult mouse has been determined by radioimmunoassay. Sympathectomy did not reduce corneal substance P levels. However substance P levels were reduced by 42% following surgical denervation and by 80% following neonatal capsaicin treatment, suggesting that corneal substance P is contained in the peripheral terminals of small diameter sensory neurones. In some mice capsaicin treatment reduced corneal sensitivity; it also resulted in marked corneal vascularization. These effects could not be ascribed to the reduction in corneal substance P.

An outbreak of adenovirus type 4 conjunctivitis.

An unusually large outbreak of conjunctivitis caused by adenovirus type 4 involved 113 proved cases during a 7-month period. Though the disease was less severe than is commonly seen in epidemic keratoconjunctivitis caused by other serotypes, the cornea was affected in 23% of cases and symptoms persisted for at least 5 weeks in 6 patients. A concurrent increase in nonocular adenovirus type 4 infections in the Bristol area was not observed. Nine of the cases could have arisen as a result of hospital cross-infection.

An outbreak of adenovirus keratoconjunctivitis in bristol.

Nineteen cases of keratoconjunctivitis caused by an adenovirus serologically related to types 10 and 19 are described. Seventeen of the patients presented over a period of 7 weeks and included 4 who were involved in a minor outbreak at a factory. The presentation and clinical features closely resembled those caused by adenoviruses types 8 and 19. Mild to severe follicular conjunctivitis, superficial punctate keratitis, discrete subepithelial opacities, membrane formation, and conjunctival scarring were all observed.

Lamellar macular hole associated with idiopathic juxtafoveolar telangiectasia.

A case of juxtafoveolar telangiectasia complicated by lamellar, retinal hole is reported and the pathogenesis discussed. This association is not commonly recognised.

Tenascin-C expression in normal, inflamed, and scarred human corneas.

AIMS/BACKGROUND: In adult tissues the expression of tenascin-cytotactin (TN-C), an extracellular matrix glycoprotein, is limited to tumours and regions of continuous renewal. It is also transiently expressed in cutaneous and corneal wound healing. There are limited data regarding its expression in inflammation and scarring of the adult human cornea. In this study, TN-C expression patterns in normal, inflamed, and scarred human corneas have been examined. METHODS: Penetrating keratoplasty specimens were selected from cases of herpes simplex keratitis, herpes zoster ophthalmicus, rheumatoid arthritis ulceration, bacterial keratitis, rosacea keratitis, interstitial keratitis, and previous surgery so as to encompass varying degrees of active and chronic inflammation and scarring. TN-C in these and in normal corneas was immunodetected using TN2, a monoclonal antibody to human TN-C. RESULTS: There was no TN2 immunopositivity in normal corneas except at the corneoscleral interface. In pathological corneas, TN2 immunopositivity was localised in and around regions of active inflammation, fibrosis, and neovascularisation. TN2 positivity was less in acute inflammation than in active chronic inflammation. Mature, avascular scar tissue and epithelial downgrowth were TN2 negative. CONCLUSION: These results indicate that in the adult human cornea, TN-C expression is induced in regions of inflammation, fibrosis, and neovascularisation, but that expression is absent in mature, avascular scar tissue. This suggests a role for this glycoprotein in inflammation, healing, and extracellular matrix reorganisation of the cornea.