Long-term follow-up of drusen-like lesions in patients with type II mesangiocapillary glomerulonephritis.

BACKGROUND: Drusen-like lesions beneath the retina in patients with partial lipodystrophy and type II mesangiocapillary glomerulonephritis (MCGN) were first reported in 1989. This study reports the long-term follow-up of this original cohort of patients more than 10 years later. METHODS: Three patients had undergone renal transplantation. Retinopathy was graded semiquantitatively using an international classification and grading system. Progression was assessed by comparing the visual acuities and the colour, red-free and fluorescein angiographic photographs at baseline and review. RESULTS: The visual acuity in all four patients was unchanged, as were the bilateral drusen-like lesions. The retinal pigment hypertrophy at the posterior pole of two of the patients was also unchanged. There were no signs of choroidal neovascularisation or central serous retinopathy in any patient. There was no progression of retinopathy over 10 years in any patient. CONCLUSION: This study suggests that in patients with type II MCGN, (a) factors other than drusen may contribute to choroidal neovascularisation and (b) renal transplantation does not appear to increase the risk of progression of retinopathy.

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.

The 'unified hypothesis' of Geddes et al. is not supported by the data.

Inflicted head injury to the developing brain frequently results in serious disability. The pathogenesis of the neuraxial and ocular findings in infants believed to have suffered inflicted head injury remains the subject of considerable debate. Recent neuropathology studies of fatal cases of inflicted head injury and of a foetal/perinatal non-traumatic model have led to the proposal that there is a 'unified hypothesis', the essential feature of which is hypoxic brain swelling secondary to cervicomedullary injury. It has been suggested that less than violent forces may be involved and even that some cases may not be due to trauma at all. The purpose of this paper is to provide a critical review of the data upon which these suppositions are based on a background of what is already known. It is submitted that there are serious flaws in the methodology; the conclusions reached cannot logically be drawn from the data; and the 'unified hypothesis' is not supported by the evidence. On the basis of the data presented, it is also difficult to sustain the secondary hypothesis purporting to describe a minority cohort with 'infantile encephalopathy with subdural and retinal bleeding' of non-traumatic causation.

Immunolocalisation of opticin in the human eye.

AIM: To localise the recently discovered glycoprotein opticin in the adult human eye. METHODS: Polyclonal rabbit antisera were raised against two different opticin peptides. Isolated human vitreous collagen fibrils were extracted with 8 M urea and the extract analysed by SDS-PAGE and western blotting. Paraffin embedded sections from two normal eyes were subjected to immunohistochemical analysis. RESULTS: Western blot analysis of the vitreous collagen fibril extract specifically identified opticin as a 45-50 kDa component that migrated as a doublet. Opticin was especially immunolocalised to the vitreous humour where labelling was most intense in the basal and cortical vitreous gel and less intense in the central vitreous. In addition, specific staining was observed along the surfaces of adjacent basement membranes including the internal limiting membrane (ILM) and posterior capsule of the lens. In one eye, labelling was also observed on the anterior lens capsule, but no other ocular tissues were specifically labelled. A type XVIII collagen/endostatin antibody labelled several ocular tissues including the ILM and basal vitreous gel. CONCLUSION: The immunolocalisation of opticin was confined to the vitreous humour, ILM, and lens capsule. In situ hybridisation studies have previously demonstrated opticin expression by the posterior non-pigmented ciliary epithelium. Thus, the immunolocalisation data support the proposition that the non-pigmented ciliary epithelium secretes opticin into the vitreous cavity where it associates with vitreous collagen and adjacent basement membranes. The staining along the ILM suggests a role for opticin in vitreoretinal adhesion and the co-localisation of opticin with type XVIII collagen/endostatin at the ILM raises the possibility that interactions between these two molecules might contribute to vitreoretinal adhesion.

Ultrastructural morphology and expression of proteoglycans, betaig-h3, tenascin-C, fibrillin-1, and fibronectin in bullous keratopathy.

AIMS: To investigate the ultrastructural localisation of proteoglycans (PG), betaig-h3 (keratoepithelin), tenascin-C (TN-C)), fibrillin, and fibronectin in bullous keratopathy (BK) corneas. METHODS: Five corneas from cases of pseudophakic bullous keratopathy (BK) were examined by electron microscopy. PG were demonstrated using cuprolinic blue, and the proteins betaig-h3, TN-C, fibrillin, and fibronectin were immunolocalised with rabbit anti-betaig-h3, mouse anti-TN-C (BC10 and TN2), mouse anti-fibrillin-1 (MAB2502), mouse anti-fibrillin (MAB1919), and rabbit anti-fibronectin by using a standard immunogold technique. RESULTS: Epithelial cells contained numerous vacuoles. Epithelial folds and large, electron lucent subepithelial bullae were present. Basal lamina was thickened and traversed by disrupted anchoring filaments. In the stroma, interfibrillar collagen spacing was increased and abnormally large PG were present. Descemet's membrane (DM) contained lucent spaces in which there were small filaments. Keratocyte and endothelial cells contained melanin granules. A posterior collagenous layer (PCL) contained numerous microfilaments and wide spacing collagen fibres with a periodicity of 100 nm. Large quantities of abnormal PG were observed at the endothelial face of the PCL. Very strong labelling with betaig-h3 antibody was observed in the basement membrane, Bowman's layer, stroma, DM, and PCL, but not in keratocytes and endothelial cells. Strong labelling with BC10 and TN2 was seen below the epithelium, in electron lucent spaces where the hemidesmosomes were absent, in the fibrotic pannus, in parts of Bowman's layer, the stroma, and Descemet's membrane. Labelling with BC10 was stronger and more evenly distributed than with TN2. Fibrillin-1 (MAB2502) and fibrillin (MAB1919) labelling was similar to TN-C labelling. Fibrillin (MAB1919) labelling was stronger than fibrillin-1 (MAB2502) labelling. CONCLUSIONS: Immunoelectron microscopy showed precise labelling of proteins at both the cellular and the subcellular level. Expression of proteins betaig-h3, TN-C, fibrillin, and fibronectin was highly increased compared with normal cornea. In the oedematous stroma, increased collagen fibril separation may facilitate a wider distribution of some soluble proteins, such as betaig-h3, throughout stroma. The modified expression of the proteins studied in these cases of BK may be regarded as part of an injury response.

Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin?

PURPOSE: Two mutations (R555Q and R124L) in the BIGH3 gene have been described in anterior or Bowman's layer dystrophies (CDB). The clinical, molecular, and ultrastructural findings of five families with CDB was reviewed to determine whether there is a consistent genotype:phenotype correlation. METHODS: Keratoplasty tissue from each patient was examined by light and electron microscopy (LM and EM). DNA was obtained, and exons 4 and 12 of BIGH3 were analyzed by polymerase chain reaction and single-stranded conformation polymorphism/heteroduplex analysis. Abnormally migrating products were analyzed by direct sequencing. RESULTS: In two families with type I CDB (CDBI), the R124L mutation was defined. There were light and ultrastructural features of superficial granular dystrophy and atypical banding of the "rod-shaped bodies" ultrastructurally. Patients from three families with "honeycomb" dystrophy were found to carry the R555Q mutation and had characteristic features of Bowman's dystrophy type II (CDBII). CONCLUSIONS: There is a strong genotype:phenotype correlation among CBDI (R124L) and CDBII (R555Q). LM and EM findings suggest that epithelial abnormalities may underlie the pathology of both conditions. The findings clarify the confusion over classification of the Bowman's layer dystrophies.

Graft failure in human donor corneas due to transmission of herpes simplex virus.

AIM: To report the clinical consequences of contamination of human donor corneas by herpes simplex virus (HSV) in organ culture. METHODS: Two patients without previous history of ocular HSV infection underwent penetrating keratoplasty (PK), one for keratoconus and the other for Fuchs' endothelial dystrophy. One patient suffered primary graft failure while the other developed a persistent epithelial defect, ultimately resulting in graft failure. Viral culture of swabs taken from both corneas during the early postoperative period was undertaken. The failed donor corneas were examined histopathologically by immunohistochemistry (IHC) for HSV-1 antigens, transmission electron microscopy (TEM), and by polymerase chain reaction (PCR) for HSV DNA. Both failed corneas were replaced within 6 weeks of the initial surgery. The records of the fellow donor corneas were also examined for evidence of infection. RESULTS: HSV was cultured from both corneas during the early postoperative period. Histology of both donor corneas demonstrated a thickened corneal stroma with widespread necrosis of keratocytes and loss of endothelial cells. IHC showed keratocytes positive with antibodies to HSV-1 antigens. TEM demonstrated HSV-like viral particles within degenerating keratocytes. PCR performed on the failed corneal grafts was positive for HSV-1 DNA, whereas PCR performed on the excised host corneal buttons was negative in both patients. Records of the fellow donor corneas showed that one cornea was successfully transplanted into another recipient after 18 days in organ culture, whilst the other was discarded because of extensive endothelial cell necrosis noted after 15 days in organ culture. CONCLUSION: HSV within a donor cornea may cause endothelial destruction in organ culture and both primary graft failure and ulcerative keratitis after transplantation. Endothelial necrosis of a donor cornea in culture also raises the possibility of HSV infection within the fellow cornea.

A mutation within exon 14 of the TGFBI (BIGH3) gene on chromosome 5q31 causes an asymmetric, late-onset form of lattice corneal dystrophy.

PURPOSE: Two forms of autosomal-dominant lattice corneal dystrophy (LCD), types I and IIIA, have previously been shown to be caused by different mutations within the transforming growth factor, beta-induced (TGFBI) gene. A clinical and molecular analysis of three unrelated kindreds with a clinically distinct late-onset LCD was undertaken to determine whether this phenotype is also caused by mutations within the TGFBI gene. DESIGN: Experimental study. PARTICIPANTS: Thirty-two members of three kindreds with corneal dystrophy. DNA from 100 normal control subjects was used as a control population. METHODS: Members of three kindreds with LCD were examined clinically, and blood samples were taken for DNA analysis. Mutation analysis was undertaken on all individuals for the coding region of the TGFBI gene by means of polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism/heteroduplex analysis, subcloning, and sequencing. MAIN OUTCOME MEASURES: Detection of mutations within the TGFBI gene. RESULTS: Clinical examination revealed a form of LCD that was bilateral in all but one case, with onset around the fourth to fifth decade. The majority of cases showed significant asymmetry, and in one case there was evidence of onset directly after minor superficial corneal trauma. Molecular analysis in all families demonstrated sequence changes within exon 14 of the TGFBI gene on chromosome 5q31, at codon 622 in family 3, and at codon 626 in families 1 and 2, which are presumed to be responsible for the disease. CONCLUSIONS: Previously, a late-onset form of LCD, termed IIIA, was shown to be caused by a P501T mutation in exon 11 of TGFBI. The authors present the first description of mutations in exon 14 of TGFBI causing an LCD, also of late onset. Although the condition presented is morphologically and histopathologically typical of an isolated lattice dystrophy, the age of onset and clinical course is not typical of type I, III, or IIIA lattice dystrophy. This, in conjunction with recent developments in our understanding of the molecular genetics of these disorders, calls into question the usefulness and validity of the current classification of the isolated lattice dystrophies.

Deposits and proteoglycan changes in primary and recurrent granular dystrophy of the cornea.

OBJECTIVE: To investigate the origin and distribution of granular deposits in the corneas of 3 patients with granular dystrophy, 1 of whom had previously received a lamellar keratoplasty in which the granular dystrophy had recurred. METHOD: Corneal tissue from 2 patients with primary granular dystrophy (patients 1 and 2) and from a patient with recurrent granular dystrophy (patient 3) was examined. Corneal graft tissue was fixed in (1) 3% glutaraldehyde in sodium cacodylate buffer, (2) 2.5.% glutaraldehyde in sodium acetate buffer containing cuprolinic blue, and (3) 4% paraformaldehyde in phosphate-buffered saline. RESULTS: In patient 1 (aged 48 years), electron-dense granular structures were observed in epithelium, Bowman layer, and throughout the stroma. Bowman layer was absent in several places. Patient 2 (aged 78 years) showed similar features except with more deposits in the stroma. In patient 3 (aged 48 years), granular structures were heavily deposited in the epithelium; there were also some deposits in the posterior (host) stroma, some of which were associated with partially degenerated keratocytes. Bowman layer appeared normal. In all 3 patients, the intracellular or extracellular granular structures were surrounded by fine fibrillar material and abnormal proteoglycans. Electron-lucent spaces within the corneal stroma contained large quantities of abnormal proteoglycan filaments that were attached in part to collagen fibrils. CONCLUSIONS: Results from patient 3 support an epithelial origin for the deposits, presumably from keratoepithelin, aggregated with other proteins. The role of keratocytes is less clear, although the presence of deposits in the stroma of all 3 patients, some associated with keratocytes, suggests that these cells might produce granular material in addition to abnormal proteoglycans.

Tenascin-cytotactin (TN-C) variants in pseudophakic/aphakic bullous keratopathy corneas.

PURPOSE: To examine pseudophakic/aphakic bullous keratopathy (PBK/ABK) human corneas for patterns of expression of tenascincytotactin (TN-C) variants known to mediate specific cellular functions, viz. anti-adhesion (high molecular mass (M(r))) and adhesion (low/intermediate M(r)). METHODS: PBK/ABK corneas were selected to encompass only those with bullae and without inflammation, scarring or neovascularisation. Serial sections from these and normal corneas were labelled with antibodies BC-4 (recognising all TN-C variants) and BC-2 (specific for the high M(r) TN-C variant). Bound antibody was revealed with an avidin-biotin peroxidase technique. In a given pair of corneal sections, positivity with BC-4 but not BC-2 indicates localisation of low/ intermediate M(r) TN-C variants and absence of the high M(r) TN-C variant. BC-2 identifies the high M(r) variant. RESULTS: There was no immunostaining with either BC-2 or BC-4 in normal corneas except at the corneoscleral interface, where both BC-2 and BC-4 were immunolocalised. In PBK/ABK corneas, BC-2 staining was seen in 5 of 13 corneas and was restricted mainly to epithelial basement membrane (BM) overlying bullae. BC-2 did not label the stroma. BC-4 immunostaining was present in all PBK/ABK corneas and was localised in epithelial BM, both epithelial and stromal borders of bullae, pannus, endothelial BM and in oedematous stromal regions. CONCLUSIONS: TN-C variants are differentially expressed in PBK/ABK corneas. The high M(r) variant is restricted mainly to epithelial BM overlying bullae, while low/intermediate M(r) variants occur in epithelial BM, both epithelial and stromal borders of bullae, and in pannus. Given the in vitro functions of TN-C, a role for promoting epithelial dehiscence and reattachment to the substratum in PBK/ABK corneas by high and low/intermediate M(r) variants respectively is likely.

Glycoproteins of trabecular meshwork, cornea and sclera.

PURPOSE: To analyse high-molecular-weight matrix glycoproteins in trabecular meshwork, cornea and sclera using SDS/PAGE and immuno- and lectin blotting. METHOD: Extracts of normal trabecular meshwork (TM), cornea and sclera were analysed under reducing conditions on SDS/ PAGE. Western blots were stained for total protein, and major high-molecular-weight components were identified by immunoblotting with antibodies to fibronectin (FN) and type VI collagen. Lectin blotting with PSA, MPA and DSA identified some of the glycoprotein glycans. RESULTS: FN antibody bound to the 240 kDa band in TM, cornea and sclera. Type VI collagen antibody bound more strongly to one band and less so to two other bands at approximately 200 kDA in normal TM and to a ladder of bands in cornea and sclera. PSA and DSA bound at 240, 200 and 140 kDa in TM, cornea and sclera. MPA bound at 240, 200 and 140 kDa in TM and at 240, 200 and approximately 120 kDA in cornea and sclera. CONCLUSIONS: FN is a component of the band at 240 kDA in TM, cornea and sclera. Normal TM was found to contain relatively more of one of the isoforms of the alpha 3 (VI) chain whilst cornea and sclera contained all the alpha 3 (VI) isoforms. Complex N-linked bi/tri-antennary glycans were localised in FN and the alpha 1, alpha 2 and alpha 3 (VI) chains in TM, cornea and sclera. O-linked glycans (identified by MPA binding) were located in FN and alpha 3 (VI) chains of TM, cornea and sclera.

Some ultrastructural data on Microsporidium ceylonensis, a cause of corneal microsporidiosis.

Sections of corneal tissue infected with Microsporidium ceylonensis were restained or processed for electron microscopy. Confirmation was obtained that the parasite develops in macrophages and that spores are uninucleate. New information is provided that sporoblasts and spores develop synchronously within a membrane in the host cell, spores have an anisofilar polar tube of 6-10 wide coils and 2-3 narrow coils and details are given of the spore wall and internal organisation. The parasite was compared on the one hand with Encephalitozoon, which exhibits asynchronous intravacuolar development of merogonic and sporogonic stages and has spores with isofilar polar tubes and on the other hand with species reported from mammals, of which the sporogonic stages develop synchronously within sporophorous vesicles and the spores have anisofilar polar tubes. Even so, a generic emplacement could not be established. Attention is drawn to the similarities between M. ceylonensis and Nosema sp. described from the cornea of a woman in Botswana.

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.

Glycans of the trabecular meshwork in primary open angle glaucoma.

AIMS: Glycan expression was compared in glaucomatous trabecular meshwork (TM) and normal TM in order to determine any differences which may reflect pathological changes underlying primary open angle glaucoma (POAG). METHODS: Resin embedded TM from trabeculectomy specimens from 15 eyes with POAG and from 12 eyes with normal anterior segments were probed with a panel of biotinylated lectins and an avidin-peroxidase revealing system at the light microscope level. Statistical analyses were performed on the comparative staining results. RESULTS: The lectins ConA and ePHA showed strong staining in all areas of both glaucomatous and normal TM; ePHA staining of Schlemm's canal (SC) from POAG TM was significantly less than that from normal TM (ePHA-SC p = 0.04). The lectins PSA, LCA, and SNA bound moderately strongly to SC endothelium and weakly to the endothelium of the corneoscleral meshwork (CSM); glaucomatous SC endothelial binding was significantly less than that of normal SC endothelium for PSA and LCA (PSA-SC p = 0.002, LCA-SC p = 0.002). STA and DSA showed moderately strong binding while WGA, ECA, AHA, and MPA bound weakly throughout the TM; for DSA and MPA this staining was significantly greater in POAG than in normal TM (DSA-SC p = 0.001, DSA-CSM p = 0.002, MPA-SC p = 0.01, MPA-CSM p = 0.02). Jac stained strongly throughout the TM and showed no significant difference in POAG compared with normal TM (Jac-SC p = 0.6, Jac-CSM p = 1). 1PHA, SBA, DBA, CTA, UEA-1 and LTA did not bind to glaucomatous TM or normal TM. There were no age-related changes seen. CONCLUSIONS: The expression of some complex and hybrid, bisected and non-bisected N-linked glycans is significantly diminished in glaucomatous TM compared with normal TM. Some glycans with multiple N-acetylglucosamine residues and O-linked glycans with terminal and subterminal galactosyl groups are significantly increased in POAG TM. Glycan expression does not change significantly with age in POAG or normal TM.