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Biomedical subjects

J G Crowston

Publications and source records attributed to J G Crowston.

At least 19 recordsLinked to original sources
PubMed2005

Trypan blue identifies antimetabolite treatment area in trabeculectomy.

AIM: Colourless solutions of mitomycin C (MMC) and 5-fluorouracil (5-FU) are widely used during trabeculectomy to inhibit postoperative scarring. The poor visibility of these agents on the eye has several drawbacks including the inability to accurately assess the area of treatment. This study examined the utility of using trypan blue dye to colour antimetabolites used during trabeculectomy and the effect of trypan blue on antimetabolite cytotoxicity in vitro. METHODS: For in vitro experiments, MMC (0.4 mg/ml) and 5-FU (25 mg/ml) were reconstituted with or without trypan blue. A lactate dehydrogenase release assay was used to measure drug induced cell death and viable cell number 7 days after treatment. For clinical assessment, trypan blue 0.1% was added to MMC and 5-FU to final concentrations of between 0.01% and 0.05%. The mixture was applied to Tenon's capsule and sclera via pre-wet or into dry 5x8 mm sponges (MMC and 5-FU) for 3 minutes or by direct subconjunctival injection after completion of surgery (5-FU). Twenty two consecutive patients undergoing trabeculectomy either with or without trypan blue were followed for 2 years postoperatively. RESULTS: The addition of 0.05% trypan blue to MMC or 5-FU did not alter MMC induced cell death or the number of viable fibroblast in vitro. In vivo, trypan blue clearly delineated the antimetabolite treatment area and facilitated control of excess antimetabolite at the wound margins as well as sponge removal. With direct subconjunctival injection, total staining area varied for a given volume with location of the needle tip. Any leakage from the injection site could be easily seen. No adverse effects attributable to trypan blue were found in 2 years of follow up. CONCLUSIONS: Trypan blue permits delineation of antimetabolite/tissue interactions without affecting cytoxicity for the assays investigated. Trypan blue can be used to visualise antimetabolite soaked sponges, estimate treatment area, and show areas of unintended tissue contact during trabeculectomy. The addition of trypan blue to antimetabolites has potential benefits in clinical, research, and teaching aspects of ocular surgery and therapy.

Aged↗
PubMed2004

Evaluating clinical signs in trabeculectomized eyes.

AIM: To evaluate interobserver agreement for clinical signs in trabeculectomized eyes when examined face-to-face with slit-lamp biomicroscopy (SL) or by remote examination using telemedicine (real-time remote video imaging; TM). METHOD: A system for examining trabeculectomized eyes was devised and validated. A prospective randomized interobserver agreement study was then undertaken to compare standard SL biomicroscopy and TM. Remote examination was performed using a 384 kbps Sony 5100 videoconferencing system. Three ophthalmologists each examined 40 eyes of 40 patients, who had previously undergone trabeculectomy. In rotation, two examiners used SL biomicroscopy. The third examined the eye remotely by TM. Analysis was performed to determine the variability in clinical signs and the presence or absence of systematic bias between ophthalmologists and examination methods. RESULTS: High levels of agreement were observed for paired examinations by SL biomicroscopy (SL/SL) for bleb vascularity (score range 0-10) with no systematic bias. Paired examination by SL and TM (SL/TM) also showed good levels of agreement for bleb vascularity, although the spread of disagreement was wider (95% limits of agreement 2.57 vs 2.98 (P=0.054)). For anterior chamber depth, observers agreed within +/- 10% of anterior chamber depth for 68% of eyes (SL/SL) and 51% of eyes (SL/TM) (P=0.68). Agreement was 'good' for wall thickness (kappa=0.63 +/- 0.08), bleb height (kappa=0.67 +/- 0.1), and the existence of bleb leak (kappa=0.63 +/- 0.19), but poor for bleb morphology (kappa=0.26 +/- 0.12). For the SL/TM comparison, agreement was fair for wall thickness (kappa=0.39 +/- 0.13), poor for bleb height (kappa=0.17 +/- 0.12), good for bleb leak (kappa=0.56 +/- 0.19), and fair for bleb morphology (kappa=0.31 +/- 0.12). Microcysts were not reliably detected using either technique. CONCLUSION: SL biomicroscopy and TM telemedicine examination may permit reliable clinical assessment of trabeculectomized eyes. However, remote examination with TM is more limited with respect to assessing bleb height and bleb wall thickness. The assessment of bleb morphology and microcysts was unreliable with both instruments. We propose that TM examination of trabeculectomized eyes appears safe and appropriate in situations where face-to-face examination by an ophthalmologist is not practical.

Anterior Chamber↗
PubMed2004

A pilot study of a system for grading of drainage blebs after glaucoma surgery.

PURPOSE: To develop and evaluate a novel bleb grading scheme for clinical and photographic evaluation. METHOD: A system for grading bleb photographs using widely applicable parameters was designed, and reference color photographs printed. A prospective masked agreement study was undertaken comparing slit lamp examination with mono and stereo photographs; 36 eyes of 28 patients with previous glaucoma surgery were graded according to defined parameters on a 1 to 10 scale clinically at the slit lamp by four ophthalmologists and two optometrists. Standardized stereo and mono photographs of the blebs were taken on the same day. The photographs were graded at least one week later in a masked fashion by the same observers, with grading of mono and stereo photographs also separated by one week. Analysis was performed to determine the variability and agreement between slit lamp results and photographic results, and to identify the presence of systematic bias. RESULTS: High levels of agreement were found between slit lamp and both stereo and mono photographs for vascularity indices, bleb wall thickness, and bleb elevation. Lower levels of agreement were found for the relative components of demarcated versus diffuse areas of the bleb, and for the total width of the bleb. The interquartile range for the median difference between slit lamp and photograph grading was -1.0 to 1.0 for all criteria except diffuse component (-2.0 to 2.0), and the median difference for all scores was 0.0. The median interobserver difference for all criteria was 0.0; the quartile range for all scores was between -0.5 and 1.0 except for diffuse component and width assessments whose quartiles fell in the -1.75 to 1.0 range. Examiners agreed with photographic grading within +/- 1 in more than 80% of gradings for vascularity and bleb height, within +/- 1 in more than 75% of gradings for bleb wall thickness, within +/- 2 in 61% of bleb width assessments, and +/- 2 in 59% of diffuse component. CONCLUSION: This bleb grading system is reproducible clinically and photographically. High levels of agreement between scores for photographs versus slit lamp examination were found for most categories, with good interobserver agreement for both photograph and slit lamp grading. Further refinement of scoring and reference photographs is required for optimization, especially for grading of bleb morphology.

Drainage↗
PubMed2004

T lymphocyte mediated lysis of mitomycin C treated Tenon's capsule fibroblasts.

AIMS: To evaluate the effect of T cell co-culture on mitomycin C treated and untreated Tenon's capsule fibroblasts. METHODS: IL-2 dependent allogeneic T cells were incubated over a monolayer of mitomycin C treated or control fibroblasts. Fibroblast numbers were evaluated by direct counts using phase contrast microscopy. To determine whether T cell mediated lysis was a consequence of MHC mismatch, co-culture experiments were repeated with autologous T cells. The effect of Fas receptor blockade was established by co-incubation with a Fas blocking (M3) antibody. RESULTS: T cell co-culture resulted in a dramatic reduction in fibroblast survival compared to mitomycin C treatment alone (p = 0.032). T cell killing required fibroblast/lymphocyte cell to cell contact and was observed in both allogeneic and autologous co-culture experiments. Fas blocking antibodies did not significantly inhibit T cell killing (p = 0.39). CONCLUSION: T cells augment mitomycin C treated fibroblast death in vitro. Similar mechanisms may contribute to the cytotoxic effect of mitomycin C in vivo and account for the largely hypocellular drainage blebs that are observed clinically.

Antibiotics, Antineoplastic↗
PubMed2004

The effects of single doses of beta radiation on the wound healing behaviour of human Tenon's capsule fibroblasts.

AIM: To determine the effects of single doses of beta radiation on the wound healing functions of human Tenon's capsule fibroblasts (hTf). METHODS: hTf were grown in tissue culture and irradiated with beta radiation using a strontium 90 source. The effects of beta radiation on fibroblast migration was studied using microporous transwell membranes. The effects of radiation on fibroblast contraction was investigated using a fibroblast populated collagen gels model. Production of extracellular matrix molecules (collagen I, collagen III, and fibronectin) by monolayers of irradiated fibroblasts was quantified for 14 days following single doses of beta radiation. RESULTS: Growth inhibiting doses of beta radiation did not inhibit fibroblast migration or contraction at any time point. Levels of soluble fibronectin from irradiated populations were significantly reduced after >500 cGy beta radiation. Collagen I and III levels were not reduced after any dose of radiation, and increased following treatment with 1000 cGy beta radiation. CONCLUSIONS: Growth arresting doses of beta radiation have unique effects on the wound healing behaviour of human Tenon's capsule fibroblasts. There was no significant effect on cellular migration or contraction, but ECM production was altered. Fibronectin production was inhibited following higher radiation doses, and collagen I and III production increased after 1000 cGy. The effects of single doses of beta radiation on ocular fibroblast wound healing behaviour are very different from those of 5-fluorouracil and mitomycin C, and these differences may be exploited clinically in the regulation of wound healing after glaucoma filtration surgery.

Beta Particles↗
PubMed2004

The effect of optic disc diameter on vertical cup to disc ratio percentiles in a population based cohort: the Blue Mountains Eye Study.

OBJECTIVE: The 97.5th percentile for vertical cup to disc ratio (VCDR) has been proposed as a useful tool to assist in the diagnosis of glaucoma in population studies. Previous reports of VCDR percentiles have either not been adjusted for disc size or have been calculated by regression analysis from small hospital based cohorts. The authors' aim was to generate VCDR percentiles in a large, population based sample. METHODS: Data were collected from 3654 individuals, aged 49 years or older, living in the Blue Mountains, west of Sydney. Vertical disc diameter and VCDR were determined by planimetry from stereo optic disc photographs. The distribution of VCDR and percentiles (95th, 97.5th, 99th) were calculated. RESULTS: 6678 eyes were included in the analysis. Median cup to disc ratio, 95th, 97.5th, and 99th percentile increased with vertical optic disc diameter in a linear fashion. An increase of 0.2 in median VCDR (0.35 to 0.55) was observed between small (1.1-1.3 mm) and large (1.8-2.0 mm) optic discs. An equivalent increase of 0.2 (0.59 to 0.74) was observed for the 97.5th percentile from small to large discs. CONCLUSION: VCDR percentiles for a "normal" population, adjusted for vertical optic disc diameter are presented. One quarter of all discs fell within the small or large disc categories highlighting the importance for estimating optic disc size. These data may assist in the diagnosis of glaucoma in clinical practice as well as providing a normative database. Sole use of VCDR percentile cut offs in defining glaucoma cases in population surveys requires further validation.

Fundus Oculi↗
PubMed2004

MMP inhibition prevents human lens epithelial cell migration and contraction of the lens capsule.

PURPOSE: The development of posterior capsule contraction following cataract surgery is caused by the activity of residual lens epithelial cells. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes, which are essential for cell migration and cell mediated contraction following wound healing. The authors investigated whether inhibiting MMP activity can reduce lens epithelial cell migration and as a result, lead to a reduction in cell mediated capsule contraction. METHODS: Human donor lens capsules were cultured and treated with a broad spectrum MMP inhibitor, Ilomastat (GM6001). MMP-2 and MMP-9 production were determined by ELISA. Cell migration onto the posterior capsule and capsule contraction were digitally measured. RESULTS: MMP inhibition significantly reduced lens epithelial cell migration onto the posterior capsule (p<0.05), and a reduction in capsule contraction was observed (p<0.05). CONCLUSIONS: Ilomastat significantly reduced lens epithelial cell migration onto the posterior capsule surface and inhibited capsule contraction. MMP inhibition may have a role in the therapeutic treatment of posterior capsule opacification.

Cataract Extraction↗
PubMed2002

In vivo production of interferon beta by human Tenon's fibroblasts; a possible mediator for the development of chronic conjunctival inflammation.

BACKGROUND: Chronic inflammation may develop from failure of the immune system to deactivate itself during resolution of the wound healing response, and is recognised as a major risk factor for trabeculectomy failure. Fibroblast/T cell interactions may contribute to aggressive scarring. Our previous research showed that in vitro human Tenon's fibroblast produced interferon beta was responsible for preventing T cell apoptosis, suggesting that this interaction could contribute to the development of chronic inflammation. METHODS: Immunohistological techniques were used to investigate the in vivo components of this particular fibroblast/T cell interaction in conjunctival biopsies from glaucoma patients undergoing filtration surgery. RESULTS: Fibroblast produced interferon beta and T lymphocytes were identified in human conjunctiva. CONCLUSION: The components of fibroblast mediated prevention of T cell apoptosis were identified in vivo, suggesting that the development of this interaction is possible and that it may contribute to the development of chronic inflammation and excessive scarring.

Aged↗
PubMed2001

Human tenon's fibroblast-produced ifnbeta and the prevention of t-cell apoptosis.

PURPOSE: Fibroblast-T-cell interactions may contribute to the development of chronic inflammation, a risk factor for trabeculectomy failure. This study was undertaken to determine whether normal and growth-arrested human Tenon's fibroblasts (HTF) can prevent cytokine deprivation-mediated T-cell apoptosis through the secretion of interferon (IFN)beta. METHODS: HTF were used either untreated or pretreated with mitomycin-C (MMC; 0.1 or 0.4 mg/ml) or 5-fluorouracil (5FU; 25 or 50 mg/ml). IL2-deprived T cells were cocultured with HTF. T-cell viability was measured at specific time points. Human Tenon's fibroblast-conditioned medium was used either untreated or treated with a neutralizing antibody against IFNbeta to block its action, after which IL2-deprived T cells were added and T-cell viability was measured. An image analysis system was used to determine the production of IFNbeta by either untreated or MMC-treated HTF. RESULTS: T-cell viability was significantly greater when T cells were cocultured with both untreated and growth-arrested HTF than when T cells were cultured alone (day 7, P = 0.0001). Neutralizing the action of IFNbeta blocked HTF-mediated T-cell rescue from apoptosis. Both untreated and growth-arrested HTF secrete IFNbeta, and MMC at 0.4 mg/ml appeared to increase IFNbeta production. CONCLUSIONS: Cytokine deprivation-mediated T-cell apoptosis can be prevented by the action of IFNbeta secreted by both normal and growth-arrested HTF, which suggests that growth-arrested HTF can still participate in an aggressive wound-healing reaction by mediating a persistent inflammatory phase. This may partly explain why some trabeculectomies fail in high-risk patients, despite the use of antimetabolites.

Apoptosis↗
PubMed2000

An optometrist's role of co-management in a hospital glaucoma clinic.

In recent years, optometrists have started to work in some clinical roles in hospital medical clinics. This paper outlines the current working practice in a typical glaucoma clinic at Moorfields Eye Hospital NHS Trust and goes on to establish the ability of an optometrist working with a team of ophthalmologists. In this study, 54 patients (n = 108 eyes) were recruited and clinically assessed by an optometrist. Subsequently, a research fellow (i.e. ophthalmologist) assessed the same patients independently. The results were compared in order to determine the accuracy of the optometrist's evaluation and proposed management. This paper considers the costs of employing personnel by reviewing the salaries of optometrists and medical assistants. Although nurses have started to work alongside ophthalmologists, and orthoptists may be considering such work, it is not within the scope of this paper to consider either of these groups. If optometrists are to become an accepted part of a glaucoma clinical team, consideration needs to be given to the training and experience they receive.

Clinical Competence↗
PubMed1999

Effects of antimetabolite induced cellular growth arrest on fibroblast-fibroblast interactions.

The use of single five-minute applications of antimetabolites during glaucoma filtration surgery has significantly reduced the occurrence of post-operative scarring and bleb failure. However, surgery for some patients is still unsuccessful, despite the use of antiproliferative agents, due to formation of scar tissue at the drainage site. It is not known if cells growth arrested in the treated area with a single application of antimetabolites influence the activity of adjacent non-treated cells. We hypothesise that the activity of non-treated cells recruited to the wound site may be involved in post-operative scarring. The aim of this study was to investigate the effects of antimetabolite induced cellular growth arrest on cell-cell interactions using in vitro techniques.Tenon's capsule fibroblast cultures were growth arrested by exposure for 5 minutes to mitomycin-C (0.001, 0.01 and 0.1 mg ml-1), 5-fluorouracil (0.25, 2.5 and 25 mg ml-1) or phosphate buffered saline (PBS). Following a period of serum-starvation, conditioned media (CM) were subsequently collected from the cells at intervals up to 29 days post-treatment. Correction for cell number was made prior to supplementation of serum-free medium with CM. CM were assessed for ability to support or inhibit normal non-treated fibroblast proliferation, migration and collagen contraction. Conditioned media collected from cells growth arrested with MMC or 5FU stimulated normal fibroblast proliferation, migration and collagen contraction in excess of non-conditioned serum-free medium. Peaks of fibroblast activity in CM differed according to which drug and concentration had originally been given to the treated cells. This study has demonstrated that CM collected from fibroblasts treated for 5 minutes with a range of concentrations of antimetabolites can differentially influence normal non-treated fibroblast activity. This in vitro data suggests that despite entering growth arrest, fibroblasts may still influence the behaviour of other cells via soluble mediators. They may have implications in the clinical setting, in that it may not be sufficient to suppress proliferation alone to prevent fibroblast behaviour associated with scarring after glaucoma filtration surgery.

Antimetabolites↗
PubMed1998

Understanding and controlling the scarring response: the contribution of histology and microscopy.

In response to injury, the body usually initiates a full and swift wound healing response resulting in reconstructed, repaired tissue. In certain instances, due to a variety of factors, this may not happen, an example being chronic granulating venous leg ulcers. At the other extreme, the wound may heal excessively, producing disabling hypertrophic scarring such as can occur following large, deep burn injuries. Our group is interested in the surgical treatment of the eye disease glaucoma. As will be explained, the successful surgical treatment of this disease depends on a reduced scarring response at the end of wound healing. The purpose of this article is to give an overview of our microscopic and histological experimental work which has furthered our understanding of tissue repair, particularly the scarring response and its potential modification for successful glaucoma surgery.

Antimetabolites↗
PubMed1998

Long term growth arrest of human Tenon's fibroblasts following single applications of beta radiation.

AIMS/BACKGROUND: Antimetabolites are increasingly used to manipulate the healing response after filtration surgery, but problems with thin cystic blebs have been encountered with the liquid agents commonly used such as 5-fluorouracil and mitomycin C. beta Radiation appears to be a useful adjuvant treatment for preventing scarring after trabeculectomy, resulting in diffuse rather than cystic bleb formation, but much of the basic cell biology of the ocular fibroblast response to beta radiation remains unclear. The effects of beta radiation on ocular fibroblast proliferation and cell cycling were investigated to determine the nature and duration of these effects on these cells. METHODS: In vitro cell culture techniques were used to investigate fibroblast proliferation. Cell viability was studied using trypan blue dye exclusion. The effect of radiation on cell cycling was investigated using bromodeoxyuridine uptake. p53 expression was demonstrated using immunocytochemistry. RESULTS: beta Radiation inhibited fibroblast proliferation in a dose dependent manner. Early cell death was not a prominent feature, but irradiated fibroblasts demonstrated a rapid onset and sustained period of growth arrest. p53 expression was found to be increased in irradiated cells. CONCLUSIONS: Single doses of beta radiation significantly inhibit Tenon's capsule fibroblast proliferation in vitro over a 28 day period. This inhibition is the result of a rapid onset and sustained period of growth arrest in irradiated cells. Irradiated fibroblasts show an increase in p53 expression, a nuclear phosphoprotein which has been associated with control of the cell cycle. Single applications of beta radiation may be an effective treatment for the prevention of bleb failure as a result of prolonged growth arrest of Tenon's capsule fibroblasts.

Beta Particles↗
PubMed1998

Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts.

PURPOSE: To determine whether treatment with mitomycin-c and 5-fluorouracil induces apoptotic death in cultured subconjunctival fibroblasts. METHODS: Cultured human subconjunctival Tenon's capsule fibroblasts were exposed to 5-minute applications of mitomycin-C (up to 1 mg/ml) or 5-fluorouracil (up to 50 mg/ml) or phosphate-buffered saline solution (PBS). Fibroblast apoptosis was determined by cell morphology, apoptosis-specific protein expression, and DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL). In addition, apoptosis was quantified by direct cell counts based on morphology or lactate dehydrogenase release. RESULTS: Morphologic changes characteristic of apoptosis included nuclear and cytoplasmic condensation and occasional nuclear fragmentation while the plasma membrane remained intact. Apoptosis-specific protein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibroblasts. The amount of apoptosis induced was dose dependent and partially inhibited by the addition of fetal calf serum to growth medium immediately after treatment. CONCLUSIONS: Mitomycin-C and high-dose 5-fluorouracil induce apoptosis in cultured Tenon's fibroblasts. Mitomycin-C-induced apoptosis is inhibited by fetal calf serum, indicating that exogenous factors influence the susceptibility of a fibroblast population to apoptosis. The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.

Antimetabolites↗
PubMed1997

T-lymphocyte-fibroblast interactions.

At the end of the immune response, activated T-cells are cleared by apoptosis. T-cell apoptosis induced by cytokine deprivation can be inhibited by the addition of exogenous cytokines or by a fibroblast-derived survival factor. Under normal circumstances, fibroblast-mediated T-cell survival may allow persistence of a small number of primed T-cells in tissues, which can be reactivated to initiate a secondary immune response. In abnormal situations, fibroblast-mediated T-cell survival may lead to the persistence of large numbers of T-cells producing a chronic inflammatory state. Evidence derived from wound healing suggests that a bidirectional interaction is possible, and T-cells are also capable of regulating fibroblast behaviour. Persistent T-cells in the wound site thus prolong a scarring response. Potential manipulation of these interactions would provide a novel strategy for developing new therapeutic interventions. For example, administration of the fibroblast survival factor that inhibits T-cell apoptosis may prolong lymphocyte survival in lymphopenic states such as AIDS. Inhibition of the survival factor on the other hand not only has potential in the treatment of chronic inflammatory states, but may also be of value in regulating scar formation with implications for the treatment of many diseases.

Animals↗
PubMed1994

Ophthalmologic features of thallium poisoning.

Thallium intoxication is characterized by the development of painful peripheral neuropathy, alopecia, mental disorders, and in severe cases, respiratory failure and death. Toxic optic neuropathy is also a feature. Ophthalmologic features of thallium poisoning include optic neuropathy, blepharoptosis, lens opacities, and ophthalmoplegia. A 44-year-old man with criminal sublethal thallium poisoning was examined one month after he was seen in the neurology department with classic systemic features. He was found to have diminished contrast sensitivity, a tritan defect in color vision, and a relative cecocentral scotoma before he developed optic atrophy.

Adult↗