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.

Lectin histochemistry of cerebral microvessels in ageing, Alzheimer's disease and Down's syndrome.

A panel of 14 lectins was used to investigate the expression of saccharides by cerebral microvessels (MBV) in ageing, Alzheimer's disease (AD) and Down's syndrome (DS). Broad increases in lectin binding with age may reflect changes in amount and diversity of glycoproteins due to the thickening of the basement membrane (BM) common in older persons. In AD, and in persons over 50 years of age with DS, binding of e-PHA, 1-PHA and PAA was increased beyond that of age alone, as was that of UEA-I and BSA-1B4 in AD, but not in DS. Persons under 50 years of age with DS showed no changes inappropriate to their age. These specific increases in AD and DS may reflect selective disease-related changes in BM and could indicate an impaired blood-brain barrier (bbb) function or integrity. However, because they occur (in DS) after the deposition of amyloid (A4) protein and onset of neurofibrillary degeneration, it is unlikely they induce plaque and tangle formation. Such changes in MBV could stem from the loss of neurones from locus caeruleus, raphe and nucleus basalis (which are thought to innervate MBV and exert control over blood flow and permeability) that occurs in DS after 50 years of age.

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.

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.

Saccharides of senile plaques and neurofibrillary tangles in Alzheimer's disease.

The contribution that oligosaccharides might make to the structure of the senile plaque and the neurofibrillary tangle was investigated using lectin histochemistry in 9 patients with Alzheimer's disease. One group of 4 lectins diffusely stained the neurites of senile plaques whereas two groups of 6 different lectins stained neurofibrillary tangles within neuronal perikarya and plaque neurites. Neuraminidase pretreatment abolished staining of tangles by one of these latter groups, but did not affect staining by the other group. Senile plaque core amyloid failed to stain with any lectin. It is concluded that oligosaccharides may contribute, but in different ways, to glycoprotein or glycolipid residues that form an integral part of the structure of the senile plaque and the neurofibrillary tangle.

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.

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.

Ophthalmic molluscum contagiosum: clinical and immunopathological features.

AIMS: A study of ophthalmic molluscum contagiosum infection was undertaken to define its clinical presentation and immunopathological features. METHODS: Retrospective analysis of 35 cases of histologically proved molluscum contagiosum infection was carried out. Diagnosis was delayed in 40% of cases resulting in repeated clinic visits. RESULTS: Twenty one patients were noted to have ocular surface changes; two patients were immunocompromised. All cases were eventually treated by excision of the lesion (19 had cautery to the lesion base) and there was recurrence in two cases. Immunohistochemical analysis of biopsy specimens showed that T lymphocytes were a consistent finding in adjacent dermis and epidermis although they did not infiltrate the molluscum lesions. Smaller numbers of macrophages were also demonstrated. There was a notable cross reactivity by a T cell antibody to the molluscum bodies. CONCLUSION: The study shows the varied presentation of molluscum contagiosum infection to the ophthalmologist and the nature of the local immune response to the virus.

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.

Lectin binding sites in normal, scarred, and lattice dystrophy corneas.

Normal, scarred, and dystrophic corneas were histochemically probed with a panel of 16 lectins by means of an avidin-biotin revealing system. Normal corneal epithelial cells, keratocytes, and endothelial cells expressed at least two distinct N-linked oligosaccharide subsets, of the non-bisected, biantennary and bisected, bi-/triantennary types. Corneal scars stained variably with the lectin subsets described above, and with Maclura pomifera agglutinin. Lattice dystrophy corneas showed a loss of the oligosaccharide expression observed on the plasma membranes of normal epithelial cells, and there was concurrent deposition of extracellular glycoprotein within the corneal stroma, which was of the same oligosaccharide subsets as were lost from the epithelial cell plasma membranes. This extracellular stromal glycoprotein was far more widely deposited than the amyloid and extended well beyond the stromal scarring. We propose that these observations are related and that in lattice corneal dystrophy a glycoprotein(s) is shed from the plasma membranes of epithelial cells and sequestrated within the corneal stroma, where it subsequently stimulates amyloid deposition.

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.

Localisation of alpha(2,3) and alpha(2,6) linked terminal sialic acid groups in human trabecular meshwork.

Sialic acid specific lectins were used to localise isomers of sialyl glycosides in human trabecular meshwork (TM) at the ultrastructural level. A lectin immunogold method demonstrated that sialic groups were concentrated on the endothelial surface of Schlemm's canal (SC) and in the adjacent juxta-canalicular tissue (JCT). One sialyl glycoside, alpha(2,6) linked N-acetyl neuraminic acid, was present mainly on the luminal aspect of the SC endothelium and in the cytoplasm of the JCT cells. Another, alpha(2,3) linked N-acetyl neuraminic acid, was localised predominantly to the extracellular fibrillar material of the JCT. The existence of a topographical segregation of these two sialyl glycosides within the TM supports the view that highly charged anionic molecules may be of significance in regulating aqueous outflow.

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.

A monoclonal antibody which discriminates between sub-types of astrocytoma.

A monoclonal antibody, M2, was produced by somatic cell hybridisation of splenocytes, from mice immunised with human fetal brain, with the murine myeloma cell line NS-1. Indirect immuno-peroxidase staining of formalin-fixed, paraffin embedded tissue sections showed that, whilst the monoclonal antibody gave a positive reaction with 32/39 astrocytomas from adult patients and 33/36 of children's astrocytomas of the adult histological type, only 17/39 of juvenile astrocytomas were stained. A Chi-squared test showed that the difference in staining between the two groups (adult versus juvenile) was highly significant (p less than 0.0001). In contrast, using a polyclonal antiserum to GFAP, a significantly larger proportion of juvenile astrocytomas than adult astrocytomas stained positively (p less than 0.05). Thus, whereas the distribution of GFAP accorded with the general finding that the degree of malignancy of a tumour correlates with the loss of cell type specific markers, the distribution of M2 reactivity was similar to that of some oncogene products which increase with malignancy. From the flow cytometry data it is apparent that the antigen recognised by M2 is not cell cycle dependent.

Corneal donor infection by herpes simplex virus: herpes simplex virus DNA in donor corneas.

Three corneoscleral discs (from two donors) underwent subtotal endothelial loss during routine "long-term" organ culture storage. Laboratory studies of these corneas revealed evidence of herpes simplex virus (HSV) infection. The fellow cornea from one of the donors had been issued for transplant to a patient with keratoconus. Deterioration of the graft was noted 5 days after surgery; the disc was removed at 2 months and was shown to be infected with HSV. In an experiment designed to simulate initial "cleansing" of donor globes, 0.1% polyvinylpyrolidone-iodine protected cells from infection with HSV. It was concluded that the detection of HSV in these corneas could not be explained by external contamination of the ocular surface. Furthermore, culture of conjunctival and pharangeal swabs taken from 47 consecutive donors confirmed that HSV is rarely isolated at or around the time of death. Five pairs of donor corneas destined for use in transplantation were selected at random and investigated for the presence of HSV. HSV DNA was detected by polymerase chain reaction (PCR) in tissue from two of the corneal donors. Sequential stepwise sectioning suggested that HSV DNA when present was distributed in discrete foci within the cornea. These observations suggest that HSV infection may be a cause of severe endothelial loss during corneal organ culture and possibly provide an explanation for some "failures" of corneal grafting.

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.