Detection of herpes simplex virus type 1, 2 and varicella zoster virus DNA in recipient corneal buttons.

AIM: To study the value of polymerase chain reaction (PCR) analysis, to detect viral DNA in recipient corneal buttons taken at the time of penetrating keratoplasty (PKP) in patients with an initial diagnosis of herpetic stromal keratitis (HSK). Since HSK has a tendency to recur, an accurate diagnosis of previous HSK could be the reason to start antiviral treatment immediately, thereby possibly decreasing the number of graft failures due to recurrent herpetic keratitis. METHODS: Recipient corneal buttons and aqueous humour (AH) samples were obtained at the time of PKP from HSK patients (n=31) and from other patients (n=78). Eye bank corneas were also used (n=23). Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and varicella zoster virus (VZV) infection were assessed by PCR and antibody detection. RESULTS: The clinical diagnosis HSK could be confirmed by PCR for HSV-1 in 10/31 (32%). In these corneal buttons HSV-2 DNA was detected in 1/31 (3%) and VZV DNA in 6/31 (19%). Intraocular anti-HSV antibody production was detected in 9/28 AH samples tested (32%). In the other patient derived corneas HSV-1 DNA was detected in 13/78 (17%), including eight failed corneal grafts without clinically obvious herpetic keratitis in the medical history. In clear eye bank corneas HSV-1 was detected in 1/23 (4%). CONCLUSIONS: PCR of HSV-1 on corneal buttons can be a useful diagnostic tool in addition to detection of intraocular anti-HSV antibody production. Furthermore, the results were suggestive for the involvement of corneal HSV infection during allograft failure of corneas without previous clinical characteristic signs of herpetic keratitis.

Long term follow up of premature infants: detection of strabismus, amblyopia, and refractive errors.

AIM: To establish recommendations for long term ophthalmological follow up of prematurely born infants. METHODS: 130 infants with a gestational age (GA) <37 weeks and born between 1 November 1989 and 31 October 1990 were enrolled in a prospective study about the development of strabismus, amblyopia, and refractive errors. Infants were subdivided in three groups according to GA: A <28 weeks (n=32), B >/=28-32-<37 weeks (n=34). Ophthalmological assessment took place at the postconceptional age of 32 weeks, at term and at 3, 6, 12, and 30 months post term. At the age of 5 years parents received a questionnaire and a majority of the children was examined again (n=99). RESULTS: At the age of 5 years 46 infants were known to have strabismus (n=29) and/or amblyopia (n=22) and/or refractive errors (n=22). Statistical analysis showed that gestational age, duration of supplementary oxygen, and duration of hospitalisation were important predictive variables for the development of strabismus, amblyopia, or refractive errors (SAR) at the age of 5 years (p<0.05). Infants with a GA 32 weeks, who developed an incidence comparable with the normal population. Strabismus developed mainly in the first year of life and at the age of 5 years. Most infants with amblyopia were detected at the age of 2-3 years. Refractive errors were found in the first year of life and at the age of 2.5 and 5 years. CONCLUSION: Infants with a GA <32 weeks should be selected for long term ophthalmological follow up. These infants should be screened at the age of 1 year, in the third year of life (preferably at 30 months), and just before school age (including testing of visual acuity with optotypes).

Cytokines in aqueous humour and serum before and after corneal transplantation and during rejection.

Cytokine profiles in aqueous humour were studied in relation to corneal disease and subsequent corneal graft survival or rejection. Cytokine levels in samples obtained from eyes with clear grafts (n = 59) were all within the normal range. At the time of penetrating keratoplasty (n = 146), intraocular levels of IL-6 were increased in 38% (50/131), most markedly in eyes with previous allograft failure or herpetic stromal keratitis. The level of IL-10 was increased in 1 eye (n = 144) and of IL-4 and IFN-gamma in none. During rejection (n = 10), the levels of IL-6 in aqueous humour were increased in 75% (3/4), of IL-10 in 50% (3/6), of IL-4 in none (0/4) and of IFN-gamma in 40% (2/5). In conclusion, the levels of total protein and IL-6 were increased prior to penetrating keratoplasty in eyes with previous inflammation. These results could however not predict the final outcome of the graft. Increased intraocular levels of IL-6, IL-10 and IFN-gamma were observed during rejection.

Are cytokine patterns in aqueous humour useful in distinguishing corneal graft rejection from opacification due to herpetic stromal keratitis?

PURPOSE: Intra-ocular cytokine profiles were determined to study the immunological mechanisms of corneal graft opacification due to rejection and/or herpetic stromal keratitis (HSK). METHODS: Sera and aqueous humour (AH) were sampled shortly after the onset of corneal graft opacification, group I (n=18). In eyes with clear grafts, samples were taken 5 months after transplantation, group II (n=59). Samples of non-inflamed eyes, prior to cataract surgery, were used to determine baseline cytokine levels, group III (n=49). Total protein (TP) levels were measured with Bradford reagent and interleukin (IL)-6, IL-10, IL-4 and interferon (IFN)-gamma with ELISAs. RESULTS: All patients who's corneal grafts showed clinical evidence of graft opacification due to rejection and/or HSK were sampled. In the AH-samples of group I, increased levels of TP were found in 60% (9/15), IL-6 in 79% (11/14), IL-10 in 39% (7/18) and IL-4 in none (0/12). IFN-gamma was detected in 19% (3/16), in the case of HSK only. In contrast, samples obtained from patients with clear grafts in group II showed increased levels of TP in 36% (20/55), IL-6 in 14% (8/57) and IL-10, IL-4 or IFN-gamma in none (n=58). CONCLUSIONS: During corneal graft rejection and/or HSV-infection, increased levels of TP and IL-6 in AH confirmed anterior chamber inflammation with breakdown of the blood-aqueous barrier. Based on the data presented, cytokine patterns in the AH do not appear to distinguish corneal opacification due to graft rejection from that due to herpes keratitis.

Development of visual function in hemihydranencephaly.

This study reports on the findings of longitudinal follow-up of visual function in a 12-year-old girl affected by congenital right hemihydranencephaly. This extremely rare unilateral brain malformation allowed the authors to gather new information on neuronal plasticity and functional compensations of the visual system across a period of 10 years. An extension of the preserved right visual hemifields above the middle line and strategical eye or head positions developed to increase visual functions are discussed. In addition, ophthalmological and orthoptical findings, as well as the development of monocular grating and linear acuity, are described.

Visual behaviours of neurologically impaired children with cerebral visual impairment: an ethological study.

BACKGROUND/AIMS: Visual functions of neurologically impaired children with permanent cerebral visual impairment (CVI) can be difficult to determine. This study investigated the behavioural profile of CVI children by means of ethological observations in order to gain a better understanding of their visual functions. METHODS: Video registrations of nine subjects who were unable to undergo more orthodox methods of visual function testing were observed and analysed by an ethologist. RESULTS: A series of behaviours (direct signs) and supportive or confirming behavioural elements (indirect signs) indicating some visual perception in the children were found. CONCLUSION: Detailed ethological observations of visual behaviour were shown to be useful for analysing visual functions of children with permanent CVI.

Azithromycin for ocular toxoplasmosis.

AIMS: To investigate the efficacy of azithromycin in patients with ocular toxoplasmosis. METHODS: 11 immunocompetent patients with ocular toxoplasmosis were treated with azithromycin (500 mg the first day, followed by 250 mg/day for 5 weeks). Ocular and systemic examinations were performed during active retinitis episodes and all patients were followed for at least 1 year. RESULTS: The intraocular inflammation disappeared within 4 weeks in seven patients, including two cases with progressive retinitis despite previous treatment with pyrimethamine, sulphadiazine, and folinic acid. Recurrence of retinitis occurred in three patients (27%) within the first year of follow up. No systemic side effects of azithromycin were encountered. CONCLUSION: These results indicate that although azithromycin cannot prevent recurrent disease it may be an effective alternative for patients with ocular toxoplasmosis who cannot tolerate standard therapies.

Longitudinal follow-up of grating acuity in children affected by cerebral palsy: results of a 5 year study.

PURPOSE: To analyse the natural course of visual acuity in subjects affected by cerebral palsy. METHODS: During the past 5 years, 16 children underwent repeated grating acuity measurements using the acuity card procedure. At the end of the follow-up final grating acuity was compared with linear acuity obtained using standard optotypes. RESULTS: A good or moderate improvement in grating acuity was shown by 14 subjects. However, the general development of grating acuity showed a protracted course and early values did not correlate with final grating acuity (r = 0.20). Moreover, most of the children showed subnormal vision when measured with standard optotypes. CONCLUSION: Clinicians should remain optimistic about the potential for some visual development in children affected by cerebral palsy. However, great caution should be exercised in extrapolating information from early grating acuity measurements. Regular assessments with the acuity card procedure are necessary in order to gain an insight into the natural course of visual development in children affected by cerebral palsy.

Peripheral multifocal chorioretinitis: a distinct clinical entity?

OBJECTIVE: The purpose of the study is to delineate the clinical features, complications, visual prognosis, and associated systemic diseases of peripheral multifocal chorioretinitis. DESIGN: The study design was a retrospective study. PARTICIPANTS: Of 828 patients with uveitis, 53 patients (6.4%) fulfilled all 3 of the following criteria: (1) the presence of multiple (>10), small, round, punched-out lesions in the peripheral retina; (2) the absence of central chorioretinal lesions; and (3) an associated intraocular inflammatory reaction. RESULTS: The majority of patients were elderly white females with bilateral ocular involvement. The presenting symptoms consisted of vitreitis and/or iritis, papillitis, and numerous retinal punched-out lesions in the periphery. On initial examination, the complications included cystoid macular edema (CME) (48%), glaucoma (25%), and cataracts (19%), resulting in a mean visual acuity of 20/80. After more than 2 years of follow-up, CME was found in 72% and cataract in 62% of the affected eyes. Submacular neovascularization never developed. The final mean visual acuity was 20/60; this was mainly dependent on the presence of CME (eyes with CME; visual acuity was 20/80, eyes without CME; visual acuity was 20/50). In 25% of patients, an association with sarcoidosis was observed (histologic and radiologic diagnoses), and an additional 29% of patients had elevated serum angiotensin-converting enzyme levels. CONCLUSIONS: Within the spectrum of multifocal chorioretinitis, the authors have defined a distinct clinical entity of peripheral multifocal chorioretinitis. The recognition of this clinical entity may be valuable because of its specific symptoms, prognosis, and association with sarcoidosis.

Corneal endothelium and growth factors.

Human corneal endothelium is characterized by a low regenerative capacity, mainly because of a low mitotic activity, and therefore complete regeneration of the endothelial layer after injury is precluded. A decrease in endothelial cell density and an abnormal cell mosaic, which may occur after many conditions, can compromise the integrity of the endothelial monolayer, resulting in corneal decompensation with reduced vision and eventual need for penetrating keratoplasty. It would be beneficial to have growth factors that can help to maintain or restore the integrity of this delicate endothelial monolayer by maintaining or increasing the endothelial cell density or to stimulate the regeneration during wound healing. Growth factors represent a group of signalling peptides which influence diverse cellular processes, including cell proliferation, differentiation, migration and survival. One of the areas that has received great interest is its enhancement of wound healing. In this review the effects of three most effective growth factors (EGF, PDGF, FGF) on corneal endothelium, especially on wound healing in human corneal buttons, will be discussed.

Synergistic action of heparin and serum on basic fibroblast growth factor-modulated DNA synthesis and mitochondrial activity of cultured bovine corneal endothelial cells.

Basic fibroblast growth factor (bFGF) is a major mitogen and chemoattractant for many cell types. The synergistic role of fetal bovine serum (FBS) and heparin on the modulation of tissue-cultured bovine corneal endothelial cells by bFGF was studied. Cell modulation was assessed by DNA synthesis ([3H]thymidine incorporation), mitochondrial activity (MTT assay), mitochondrial volume (transmission electron microscope study), and cell shape in mitotically competent cells and cells mitotically inhibited by 5-fluorouracil, mitomycin C, and irradiation. The dose-dependent stimulation of bFGF was found to be different for DNA synthesis and mitochondrial activity, the maximal effective dose being 1 and 10 ng/ml, respectively. Supplementation of bFGF with FBS enhanced both DNA synthesis and mitochondrial activity and caused a shift in the dose response to lower bFGF concentrations for the DAN synthesis, but not for the mitochondrial activity. Supplementation of bFGF with heparin resulted in an additional response for the mitochondrial activity, but not for the DNA synthesis. In addition, bFGF increased mitochondrial volume and induced elongation of the cells. These processes were further enhanced by the addition of heparin to bFGF. These results suggest that serum and heparin have a differential effect on mitogenic and non-mitogenic cells processes modulated by bFGF.

Basic fibroblast growth factor stimulates corneal endothelial cell growth and endothelial wound healing of human corneas.

PURPOSE: To determine the dose response of human recombinant basic fibroblast growth factor (bFGF) on mitogenic activity, and the supplementary role of serum in cultured bovine and human corneal endothelial cells (BCECs, HCECs). To investigate the effect of bFGF on endothelial wound healing of human corneas in vitro. METHODS: In cell culture, DNA synthesis was assessed by 3H-thymidine incorporation. Wound healing was studied using paired human corneas after mechanical damaging of the endothelium. One cornea was treated with bFGF, and the mate served as control. Wound closure was determined after staining with trypan blue. Endothelial cell density (ECD) was assessed in the closed wound area after alizarin red staining. DNA synthesis was assessed using 3H-thymidine autoradiography. RESULTS: In cell culture, bFGF induced a dose-dependent mitogenic response on BCECs and HCECs. Addition of serum to the culture medium shifted the dose-response curve to considerably lower bFGF concentrations. In organ culture, the time of complete wound closure shortened only marginally (0.5 day) after bFGF treatment (P < 0.01). In the closed wound center, ECD was significantly higher in 1 ng/ml bFGF-treated corneas (686 +/- 134 cells/mm2) than in controls (554 +/- 117 cells/mm2), an increase of +25%. Doses of 0.1 and 10 ng/ml also were effective, but less so than with 1 ng/ml (+11% and +15%, respectively), whereas a dose of 100 ng/ml even had a negative effect (-11%). DNA synthesis was marginally enhanced in bFGF-treated (1 ng/ml) corneas. CONCLUSIONS: The maximal effective dose of bFGF producing a BCEC mitogenic response is dependent on serum. In human senior donor corneas, bFGF promotes endothelial wound healing predominantly by stimulation of cell migration.

Effects of platelet-derived growth factor on endothelial wound healing of human corneas.

PURPOSE: To investigate the effects of platelet-derived growth factor (PDGF) on endothelial wound healing of organ-cultured human corneas. METHODS: The endothelia of paired human donor corneas (age, 71 +/- 11 years; total 84 pairs) were mechanically wounded (area, 5.6 +/- 0.8 mm2). Of each pair, one cornea was treated with 10 ng/ml human recombinant PDGF-BB while its mate served as control. The endothelial wound closure time was assessed by daily staining of the corneas with trypan blue. Morphometric data (endothelial cell density, shape, coefficient of variations) were obtained in the wound area after alizarin red staining. DNA synthesis was assessed using 3H-thymidine autoradiography. RESULTS: Although significant, the time of complete wound closure shortened only marginally on addition of PDGF to the culture medium. In the closed wound center (between 4 and 9 days), all corneas exposed to PDGF had significantly higher endothelial cell densities (737 +/- 126 cells/mm2) than the control corneas (515 +/- 89 cells/mm2). Fifteen days after wounding, the mean endothelial cell density averaged 526 +/- 93 and 708 +/- 135 cells/mm2 in the control and PDGF-treated groups, respectively. PDGF did not affect the final cell shape within the closed wounds. DNA synthesis was significantly but only marginally enhanced in PDGF-treated corneas. CONCLUSION: In organ-cultured human corneas, PDGF-BB promotes endothelial wound healing predominantly by cell migration, at least in corneas from senior donors.

Platelet-derived growth factor: receptor expression in corneas and effects on corneal cells.

PURPOSE: Platelet-derived growth factor (PDGF), a major mitogen and chemoattractant, is a dimeric molecule of disulfide-bonded A and/or B polypeptide chains (PDGF-AA/AB/BB). Two PDGF receptors (PDGFR) exist, alpha and beta, which dimerize after ligand exposure. The alpha-receptor binds both A- and B-chains, whereas the beta-receptor preferentially binds the B-chain. Whether PDGFR are present on, and whether PDGF is mitogenic for, corneal cells was investigated. METHODS: For receptor determination, a two-step immunoperoxidase technique with monoclonal antibodies against both alpha- and beta-receptors was applied on frozen sections of human and bovine corneas. To test the mitogenic activity of PDGF-BB, two proliferation assays, the DNA synthesis assay (3H-thymidine incorporation) and the colorimetric MTT assay, were used for cultured bovine corneal endothelial cells (BCEC) and human corneal fibroblast (HCF). RESULTS: Both receptors were present on epithelial cells, stromal fibroblasts, and endothelial cells, the beta-receptor being most abundant. In BCEC, minimal and maximal effects on DNA synthesis occurred at 10 ng/ml and 50-100 ng/ml PDGF, respectively. For HCF, the minimal and maximal effective doses were 1 ng/ml and 25-100 ng/ml of PDGF, respectively. The MTT assay, carried out in BCEC only, showed a minimal and maximal cell activity at 1 ng/ml and 10-100 ng/ml of PDGF, respectively. CONCLUSIONS: The presence of PDGFR in human corneal epithelium, fibroblasts, and endothelium and the mitogenic effects of PDGF on corneal cells indicate that PDGF may play a role in corneal wound healing.

Effects of human epidermal growth factor on endothelial wound healing of human corneas.

Paired human donor corneas (age, 73 +/- 12 yr), preserved in organ culture medium, were used to evaluate the effect of human epidermal growth factor (hEGF) on endothelial wound closure rate (WCR), on morphometric parameters (cell size, shape, and density), and on cell division in the wound area. The endothelium of the corneas was mechanically wounded (area, 4.9 +/- 0.9 mm2). For each pair, one cornea was treated with 10 ng/ml hEGF, while the mate served as control. WCR was assessed by daily staining of the corneas with trypan blue. Morphometric data were obtained after alizarin staining. Mitotic activity was assessed using 3H-thymidine autoradiography. Addition of hEGF significantly increased the WCR compared to the control group. In the closed wound (between 4-9 d), the mean cell size in the center averaged 1940 microns2 in the control group and 1287 microns2 in the hEGF-treated group (P less than 0.01). Fifteen days after wounding, the mean cell sizes averaged 1910 microns2 and 1427 microns2 in the control and hEGF-treated group, respectively (P less than 0.01). All corneas exposed to hEGF had higher endothelial cell densities than the control corneas. In the early stages of wound closure, the cells in the transitional zone in hEGF-treated corneas had a somewhat more elongated shape. However, hEGF did not affect the final cell shape within the closed wound. Autoradiographic results revealed that hEGF accelerated DNA-synthesis, although only to a limited extent. The results indicate that, in human corneas, hEGF promotes endothelial wound healing predominantly by cell migration, at least in corneas from senior donors.

Human corneal endothelial wound repair. In vitro and in vivo.

Endothelial wound repair was studied in 85 organ-cultured human corneas classed in Groups I--IV by means of a combined silver stain, nuclear stain, and autoradiography after denuding the endothelium from a circular area in the center of the cornea. Endothelial migration into the defect occurred first, but by 48 hours (before the wound was completely covered), incorporation of tritiated thymidine showed synthesis of DNA had occurred. Cell division, shown by the presence of mitotic figures, was greatest at 72 hours. In Group V, the endothelial wound repair was studied in vivo in two patients (a 65-year-old and a 67-year-old). The wound was created by a central transcorneal freeze. After removing the cornea from the enucleated eye, the wound was exposed for one hour to tritiated thymidine at 37 C in vitro. The results of Group V were similar to those of Groups I--IV. Both migration and proliferation were found.

Intracapsular fixation of extracapsular pseudophakia.

A modification of the fixation of the Binkhorst iridocapsular lens is described. The elastic force of the anterior capsule is used in order to gain a stable, safe and central position of the two-loop iridocapsular lens in combination with an extracapsular cataract extraction. Miotic therapy is not necessary if one uses the described method.

Immunological activity to different corneal antigens in patients with corneal diseases.

Patients suffering from various corneal diseases and waiting for keratoplasty have been immunologically investigated in order to establish sensitisation to corneal antigens. The presence of lymphocytes sensitised to the soluble from human corneas, bovine corneal epithelium, and bovine corneal stroma, which all possess common antigenicity, could be demonstrated in 30%, 50%, and 23%, respectively, of all patients. In none of these patients could a positive plasma antibody titre to human corneal antigens be detected. The results suggest the dominance of T-lymphocyte activity. No correlation was found between the degree of corneal vascularisation and the presence of sensitised lymphocytes to human corneal antigens. Arrangement of the patients according to diagnosis showed that especially those suffering from herpes simplex virus keratitis reacted positively to human corneal antigens. A possible explanation is given. Lymphocytes of controls showed no or only very low stimulation with the soluble fractions of human corneas or bovine corneal stromas. The soluble fraction of bovine corneal epithelium stimulated the lymphocytes of 6 out of 19 controls. The elimination of the donor corneal epithelium before transplantation may be beneficial in view of the involvement of histocompatibility antigens.