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A J Quantock

Publications and source records attributed to A J Quantock.

At least 19 recordsLinked to original sources
PubMed2001

The use of X-ray scattering techniques to determine corneal ultrastructure.

The manner in which X-rays are scattered or diffracted by the cornea provides us with valuable insights into the fine structure of the corneal stroma. This is because when X-rays pass through a cornea a diffraction pattern is formed due to scattering from regularly arranged collagen molecules and fibrils that comprise the bulk of the stromal matrix. Collagen provides the cornea with most of its strength, and its proper organisation is believed to be important for tissue transparency. Ever since 1978, when the first X-ray diffraction patterns were obtained from the cornea using radiation from a powerful synchrotron source, biophysicists have recorded and analysed a huge number of X-ray diffraction patterns from many different corneas. This article aims to explain the ideas that underpin our use of X-ray diffraction to investigate corneal ultrastructure, and show how the knowledge gained to date has far-reaching implications for tissue biomechanics, disease changes and transparency.

Animals↗
PubMed2000

Ultrastructural localization of sulfated and unsulfated keratan sulfate in normal and macular corneal dystrophy type I.

Keratan sulfate (KS) proteoglycans are of importance for the maintenance of corneal transparency as evidenced in the condition macular corneal dystrophy type I (MCD I), a disorder involving the absence of KS sulfation, in which the cornea becomes opaque. In this transmission electron microscope study quantitative immuno- and histochemical methods have been used to examine a normal and MCD I cornea. The monoclonal antibody, 5-D-4, has been used to localize sulfated KS and the lectin Erythrina cristagalli agglutinin (ECA) to localize poly N -acetyllactosamine (unsulfated KS). In normal cornea high levels of sulfated KS were detected in the stroma, Bowman's layer, and Descemet's membrane and low levels in the keratocytes, epithelium and endothelium. Furthermore, in normal cornea, negligible levels of labeling were found for N -acetyllactosamine (unsulfated KS). In the MCD I cornea sulfated KS was not detected anywhere, but a specific distribution of N -acetyllactosamine (unsulfated KS) was evident: deposits found in the stroma, keratocytes, and endothelium labeled heavily as did the disrupted posterior region of Descemet's membrane. However, the actual cytoplasm of cells and the undisrupted regions of stroma revealed low levels of labeling. In conclusion, little or no unsulfated KS is present in normal cornea, but in MCD I cornea the abnormal unsulfated KS was localized in deposits and did not associate with the collagen fibrils of the corneal stroma. This study has also shown that ECA is an effective probe for unsulfated KS.

Adult↗
PubMed2000

Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits.

PURPOSE: To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. METHODS: An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. RESULTS: A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. CONCLUSION: Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.

Amnion↗
PubMed2000

Conjunctival inflammation in the chronic phase of Stevens-Johnson syndrome.

AIMS: To understand the immunopathogenesis of the corneal conjunctivalisation in Stevens-Johnson syndrome. METHODS: Conjunctivalised corneas from five patients with Stevens-Johnson syndrome were studied immunohistochemically for several cell surface antigens and two cytokines. Chemical injury specimens were also studied. RESULTS: In all cases, immunohistochemistry revealed LFA-1, CD4, CD8, and CD68 on subepithelial infiltrating cells. Also, HLA-DR and ICAM-1 were found on the surfaces of epithelial cells, subepithelial infiltrating cells, subepithelial fibroblasts, and endothelial cells in blood vessels. IFN-gamma was found in basal epithelial cells; subepithelial cells and subepithelial extracellular matrix CD19 and IL4 were not detected. CONCLUSIONS: The infiltrating cell population in the Stevens-Johnson syndrome samples includes macrophages, CD4 positive T cells, and CD8 positive T cells. The cytokine expression pattern suggests CD4 positive T cells are Th1 cells. The infiltrating cell population is similar in Stevens-Johnson syndrome and chemical injury conjunctivalised corneas.

Adult↗
PubMed2000

Growth factor mRNA and protein in preserved human amniotic membrane.

PURPOSE: To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). METHODS: RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80 degrees C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2) in preserved human corneas and in AM both with and without amniotic epithelium. RESULTS: RT-PCR revealed that human AM expresses mRNA for EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. CONCLUSIONS: Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.

Amnion↗
PubMed2000

Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane.

PURPOSE: Surgery to reconstruct the ocular surface is greatly facilitated by the use of amniotic membrane, either as a biologic drape or, more recently, as a substrate for the transplantation of cultivated corneal epithelial cells. This study was designed to compare the usefulness of intact and denuded human amniotic membranes as a substrate for corneal epithelial cell culture. METHODS: Small (3-mm-diameter) biopsy specimens of superficial cornea including epithelium were excised from the central and limbal regions in rabbits. They were cultured on human amniotic membrane with or without amniotic epithelial cells and examined by light, scanning electron, and transmission electron microscopy. RESULTS: Cellular outgrowth from the central explants (n = 10) after 14 days in culture measured 1.82 +/- 2.62 mm2 on intact amniotic membrane and 131.83 +/- 28.31 mm2 on denuded amniotic membrane. In contrast, outgrowths from the limbal explants (n = 10) at the same time measured 4.58 +/- 4.56 and 505.39 +/- 134.20 mm2 on intact and denuded amniotic membranes, respectively. The leading edges of the outgrowths on intact amniotic membrane were much less uniform than those on denuded amniotic membrane, and, in the former, corneal epithelial cells appeared to migrate over the top of amniotic epithelial cells. Limbal cells cultivated on denuded amniotic membrane formed a nicely stratified layer that adhered well to the underlying amniotic membrane. CONCLUSIONS: Denuded amniotic membrane appears to be an excellent substrate for the cultivation of corneal epithelial cells, with a view to transplantation.

3T3 Cells↗
PubMed1999

Amyloid and Pro501 Thr-mutated (beta)ig-h3 gene product colocalize in lattice corneal dystrophy type IIIA.

PURPOSE: To assess the relative distribution in the cornea of amyloid and (beta)ig-h3 gene product in lattice corneal dystrophy type IIIA (LCD-IIIA). METHODS: Serial sections from the cornea of a patient with LCD-IIIA were subjected to either Congo red staining or immunohistochemistry employing an antibody to (beta)ig-h3. Also, genomic DNA was isolated from peripheral blood and used as a template for polymerase chain reaction to amplify all exons of (beta)ig-h3. RESULTS: Exon 11 of (beta)ig-h3 was mutated (Pro501Thr). Subepithelial and intrastromal congophilic deposits exhibited a birefringency characteristic of amyloid. These regions of the tissue were also highly immunoreactive with the antibody to the (beta)ig-h3 gene product. CONCLUSION: Amyloid and Pro501Thr-mutated (beta)ig-h3 protein accumulate and colocalize in LCD-IIIA.

Aged↗
PubMed1999

Epithelial hyperproliferation and transglutaminase 1 gene expression in Stevens-Johnson syndrome conjunctiva.

In Stevens-Johnson syndrome, pathological keratinization of the ordinarily nonkeratinized corneal and conjunctival mucosal epithelia results in severe visual loss. We examined conjunctiva covering cornea in five eyes in the chronic cicatricial phase of Stevens-Johnson syndrome. Normal conjunctiva from five age-matched individuals was studied also. The number of epithelial cells in Stevens-Johnson syndrome conjunctiva that were immunoreactive with a monoclonal antibody, Ki-67, to a nuclear antigen found only in proliferating cells was greater than normal (93.8+/-19.8 cells above 100 basal cells versus 12.8+/-0.5 cells above 100 basal cells; P = 0.009). In addition, although clinical inflammation was mild, massive lymphocytic infiltration was seen in the substantia propria of conjunctiva covering cornea. In situ hybridization documented transglutaminase 1 (keratinocyte transglutaminase) mRNA in suprabasal cells of the abnormally thickened conjunctival epithelium in all Stevens-Johnson syndrome patients. In contrast, no message was detected in normal conjunctival or corneal epithelia. Transglutaminase 1 is expressed during the terminal differentiation of keratinocytes where it helps synthesize cornified cell envelopes. We speculate that in Stevens-Johnson syndrome, epithelial hyperproliferation, and transglutaminase 1 gene expression lead to the pathological keratinization of ocular surface mucosal epithelia.

Adult↗
PubMed1999

Apolipoproteins J and E co-localise with amyloid in gelatinous drop-like and lattice type I corneal dystrophies.

AIMS: Apolipoprotein J (apoJ) and apolipoprotein E (apoE) are thought to contribute to amyloid formation in patients with Alzheimer's disease. The aim of this investigation was to discover whether or not these apolipoproteins associate with corneal amyloid in gelatinous drop-like corneal dystrophy (GDCD) and lattice corneal dystrophy type I (LCD-I). METHODS: Corneas from three eyes of three patients with GDCD and one eye of one patient with LCD-I were examined immunohistochemically using antibodies against apoJ and apoE. Two normal corneas were similarly examined. Tissue sections of brain from a patient with Alzheimer's disease were used as positive controls for the antibodies. For all negative controls, mouse IgG was used instead of the primary antibody. RESULTS: Intense apoJ and apoE immunoreactivities were found in congophilic amyloid deposits in GDCD and LCD-I. These deposits were located subepithelially in GDCD, and subepithelially and intrastromally in LCD-I. In GDCD, immunostaining of subepithelial amyloid with anti-apoJ was noticeably stronger than with anti-apoE. CONCLUSIONS: As in senile plaques in brain from a patient with Alzheimer's disease, apoJ and apoE co-localise with amyloid in corneas with GDCD and LCD-I.

Aged↗
PubMed1998

Two distinct kerato-epithelin mutations in Reis-Bücklers corneal dystrophy.

PURPOSE: Two patients were diagnosed with Reis-Bücklers corneal dystrophy (RBCD), although the pattern and severity of corneal opacification differed. To see whether there was a genetic basis for these phenotypic variations, we analyzed beta ig-h3, the gene that codes for kerato-epithelin and that contains a mutation (Arg555Gln) that causes RBCD. METHODS: A 30-year-old man with honeycomb-shaped subepithelial opacities in his central cornea and a 25-year-old man with progressive subepithelial geographic opacities were both considered to have RBCD. We isolated genomic DNA from leukocytes of the two patients and their family members and screened for an Arg555Gln kerato-epithelin mutation. Then we analyzed all exons of the gene using the single-strand conformation polymorphism (SSCP) technique to search for any other kerato-epithelin mutations. RESULTS: The patient with honeycomb-shaped opacities had an Arg555Gln kerato-epithelin mutation that caused his RBCD, whereas the patient with geographic opacities did not; instead, he had a new kerato-epithelin mutation (Arg124Leu), which cosegregated with his family members. CONCLUSIONS: The variant of RBCD characterized by honeycomb-shaped opacities is caused by an Arg555Gln kerato-epithelin mutation. On the other hand, a new kerato-epithelin mutation, Arg124Leu, was found to cause the RBCD variant characterized by recurrent epithelial erosions and progressive geographic subepithelial opacification. Codon 124 is a hot spot for kerato-epithelin mutations, where the mutations responsible for three autosomal dominant corneal dystrophies--lattice type I (Arg124Cys), Avellino (Arg124His), and the variant of RBCD with geographic rather than honeycomb opacities (Arg124Leu)--are located.

Adult↗
PubMed1998

Gelatinous drop-like corneal dystrophy is not one of the beta ig-h3-mutated corneal amyloidoses.

PURPOSE: To discover if beta ig-h3 is mutated in gelatinous drop-like corneal dystrophy, as has been suggested. METHODS: Genomic DNA was isolated from unrelated individuals with lattice corneal dystrophy type I (n = 3), Avellino corneal dystrophy (n = 3), and gelatinous drop-like corneal dystrophy (n = 3) and used as a template for polymerase chain reaction to amplify all exons in beta ig-h3. The polymerase chain reaction product was then sequenced. RESULTS: Beta ig-h3 is mutated in lattice corneal dystrophy type I (Arg124Cys) and Avellino corneal dystrophy (Arg124His). In gelatinous drop-like corneal dystrophy, on the other hand, no mutation was detected in the entire coding region of beta ig-h3 (all 17 exons). CONCLUSION: Unlike the amyloidotic corneal dystrophies lattice type I and Avellino, gelatinous drop-like corneal dystrophy is not likely to be caused by a mutation in beta ig-h3.

Amyloidosis↗
PubMed1998

A synchrotron x-ray diffraction study of developing chick corneas.

To study some ultrastructural aspects of developing chick corneas we performed a synchrotron x-ray diffraction analysis of 22 specimens obtained daily from developmental day 10 through day 19. Before day 12 of development in chicks we were unable to detect a meridional x-ray diffraction pattern from cornea. Neither were we able to record a first-order equatorial x-ray reflection at this time. Normally, these reflections are present in corneal x-ray patterns, arising from, respectively, the periodic axial electron density of fibrillar collagen and the lattice-like arrangement of the fibrils. By day 12 of development we could detect the third- and fifth-order meridional reflections (indicating increased amounts of collagen) and a first-order equatorial reflection (implying that more collagen was regularly arranged). The third- and fifth-order meridional reflections became more intense as the tissue matured, suggestive of a continued deposition of fibrillar collagen, and the scattering angle of the interfibrillar maximum increased, suggesting that regularly arranged collagen was becoming more closely packed with maturation. In embryonic chick corneas, the establishment of an orderly, fairly compacted matrix of collagen fibrils may be one of the main events underlying the acquisition of corneal transparency.

Animals↗
PubMed1998

Histopathology of recurrent gelatinous drop-like corneal dystrophy.

PURPOSE: To elucidate more fully the histopathology of gelatinous drop-like corneal dystrophy in a case that recurred and was operated on 7 years after the original surgery. METHODS: Transmission electron microscopy, including the use of cuprolinic blue to image sulfated proteoglycans, and horseradish peroxidase as a marker for in vitro epithelial permeability. RESULTS: Our patient's epithelium was often abnormally thick, and many intercellular spaces were present at all levels, although cell-cell contact via desmosomes was also evident. Horseradish peroxidase, when used as an in vitro tracer, was able to penetrate the most superficial tight junctions of the corneal epithelium. Basal epithelial cells were not columnar, and numerous spike-like projections protruded into the underlying amyloid/collagenous tissue from the basal epithelium. Beneath this, duplication of a discontinuous epithelial basement membrane was noted. In this region, collagen often coexisted with amyloid, the deposition of which was extensive. As in some other corneal pathologies, long-spacing collagen was detected. The association of small proteoglycans with collagen was unremarkable, although some abnormally large, sulfated proteoglycan filaments were interspersed with the amyloid and underlying stroma. CONCLUSION: Recurrent gelatinous drop-like corneal dystrophy shares several histopathologic features with its primary counterpart, although some features, such as the presence of abnormally large, sulfated proteoglycans and long-spacing collagen, the permeability of the epithelial tight junctions, and the duplication of the epithelial basement membrane, have not been reported previously.

Amyloid↗
PubMed1998

An ultrastructural investigation into proteoglycan distribution in human corneas.

PURPOSE: We report an investigation into the distribution of proteoglycans (PGs) in normal, organ-cultured and dextran-treated human corneas. METHODS: Immunogold labeling was carried out at the electron microscope level to localize keratan sulphate (KS), chondroitin sulphate (CS), and heparan sulphate (HS) PGs. RESULTS: High levels of labeling for CS was found in the epithelium, endothelium, and keratocytes, with light labelling present in the basement membranes and the corneal stroma. Labeling for HS was present in the epithelium, endothelium, and keratocytes, with intense labeling present at the endothelium/Descemet's membrane interface and the epithelium/Bowman's layer interface. Large filaments were also observed in these regions in cuprolinic blue-stained specimens. Keratan sulphate was present at high levels in the stroma and the basement membranes with low levels present within the keratocytes, epithelium, and endothelium. The pattern of KS labeling along the collagen fibrils in the stroma sometimes showed evidence of periodicity. Organ-cultured corneas had extensive collagen-free "lakes," the interior of which immunolabeled positively for KS and showed staining with cuprolinic blue. The lakes were greatly reduced in the dextran-treated samples. CONCLUSION: This investigation determined the ultrastructural distribution of KS, CS, and HS PGs in human cornea and showed that organ culture is associated with a change in distribution of stromal PGs.

Chondroitin Sulfates↗
PubMed1997

Macular corneal dystrophy type II: multiple studies on a cornea with low levels of sulphated keratan sulphate.

We investigated an individual macular corneal dystrophy (MCD) type II cornea from a 42-year-old woman with markedly reduced antigenic keratan sulphate levels. A characteristic 4.6 A X-ray reflection was evident, and the mid-stroma contained 30% less sulphur than normal. Close packing of collagen was restricted to the superficial stroma. Abnormally large proteoglycan filaments were noted throughout the extracellular matrix and Descemet's membrane's posterior non-banded zone, but not its anterior banded zone. Small, collagen-associated stromal proteoglycans were susceptible to digestion with chondroitinase ABC, but not keratanase I or N-glycanase. On occasion, collagen fibrils ranged in size from 20 nm to 58 nm, with preferential diameters of 34 nm and 42 nm. Corneal guttae were evident, as were numerous endothelial inclusions, most probably due to intracellular fibrillogranular vacuoles similar to those found in the stroma. The endothelium expressed reduced anti-keratan sulphate labelling.

Adult↗
PubMed1997

Isolation and chromosomal localization of a cornea-specific human keratin 12 gene and detection of four mutations in Meesmann corneal epithelial dystrophy.

Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I keratin.

Base Sequence↗