Views of glaucoma patients on provision of follow-up care; an assessment of patient preferences by conjoint analysis.

AIMS: To determine patients' preferences for provision of glaucoma follow-up services examining preferences for location, access and personnel for delivery of this care. METHODS: 100 patient patients attending the glaucoma outpatient clinic for follow-up review underwent an interview-based assessment during which they completed the visual function questionnaire 25 and ranking of scenario options for provision of follow-up care for their glaucoma. Percentage preferences for aspects of care offered in the conjoint analysis scenario packages and generation of utility values for each of the factor levels included in the scenario design were calculated. RESULTS: Travel time and training of health professional were the most important factors for patients (accounting for over 60%) of their preference. Utility scores were generated for each factor, with shorter travel time and examination by a doctor being the most important features to the patients. Patients who lived furthest from the hospital and had severe visual disability considered the number of visits to complete an episode to be an important feature. CONCLUSION: Patients ideally would like to travel a short distance and be seen by a doctor when being followed up for their glaucoma.

Eyetrack (ET) vs the conventional paper record (CPR): a study comparing the accuracy and speed of data retrieval from glaucoma patient records.

PURPOSE: To compare patient data retrieval between electronic patient record systems (Eyetrack) and conventional paper records (CPRs). METHODS: A total of 20 long term glaucoma patient records held on Eyetrack were randomised into two collections with 10 CPRs and 10 Eyetrack records in each collection. The Eyetrack records of one collection were the CPRs of the other collection and vice versa. Four doctors, as two groups, were assessed on a separate collection of records. The time taken to answer 10 questions and the accuracy were assessed. Comparison was made of the answers between the two formats. A month later each group was assessed on the 10 CPRs of the other collection. An expert Eyetrack user was assessed on only the 20 Eyetrack notes. Comparison was made between the 20 CPRs the doctors were assessed on and the 20 eyetrack records. RESULTS: In the first comparison, the mean time for all the doctors to answer the questions on a CPR was 324.4(+/-106.0) s compared to 104.8(+/-34.0) s for Eyetrack(Mann-Whitney, P<0.01). Mean accuracy for a CPR was 84.0%(+/-13.0%) compared to 98.0%(+/-4.0%) for Eyetrack(Mann-Whitney, P<0.01). Comparing the expert Eyetrack user with the CPR showed a mean time for Eyetrack of 96.6(+/-34.8) s compared with 283.7(+/-63.9) s for CPR(Mann-Whitney, P<0.0001). Mean accuracy for Eyetrack was 97.5%(+/-7.2%) compared to 82.0%(+/-8.7%) for CPRs(Mann-Whitney, P<0.0001). CONCLUSIONS: An improvement of 3 min 40 s per record was observed with Eyetrack. Accuracy was also improved. Similar results were also found comparing an expert Eyetrack user with CPRs.

Risk management strategies following analysis of cataract negligence claims.

INTRODUCTION: Clinical governance and risk management is very important in today's clinical practice. Cataract surgery is one of the most common procedures performed in the NHS, with around 200,000 operations per year. In order to help minimise the frequency of negligence claims, we performed a collaborative study to analyse claims relating to cataract surgery, dealt with by the defence organisations of England, Scotland, Wales, and Northern Ireland. MATERIALS AND METHODS: All claims dealt with by the Medical Defence Union, the Medical Protection Society, and the Medical and Dental Defence Union of Scotland from January 1990 to December 1999, were analysed by three ophthalmologists with at least 5 years' speciality experience. Recurrent themes were identified and claims were grouped by major causative factor. The findings were discussed by a panel compromising the authors in conjunction with the defence unions and risk management strategies were designed. RESULTS: There were 96 claims within the 10- year period analysed. Of these, the largest group (52) pertained to claims that related to accepted complications of cataract surgery. The remainder comprised two groups: 'Medical Errors' (anaesthetic, surgeon, and biometry) and 'Other Claims' comprising subjective complaints, pain and poor visual outcome. A total of 16 claims had been settled by May 2002, 45 are ongoing and 35 have closed without settlement. CONCLUSIONS: The majority of claims pertained to well-recognised complications of cataract surgery. If these risks are adequately explained to the patient before surgery and if the care provided reaches a standard acceptable to a responsible body of professional opinion, all such claims should be defensible. Good visual outcome does not protect against litigation.

A recombinogenic targeting method to modify large-inserts for cis-regulatory analysis in transgenic mice: construction and expression of a 100-kb, zebrafish Hoxa-11b-lacZ reporter gene.

The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions. In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice. PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting. A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting. Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f. transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites. These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones.

Vascular smooth muscle cell effect on endothelial cell endothelin-1 production.

Endothelin-1 (ET-1) is a potent mitogen secreted by endothelial cells (ECs) in culture and is a putative factor in vascular lesion development. The purpose of this study was to examine whether smooth muscle cells (SMCs) inhibit EC secretion of ET-1. The effect of SMCs on EC ET-1 and constitutively expressed nitric oxide (NO) synthase activity was examined by using a bilayer co-culture model. SMCs inhibited both EC ET-1 protein and RNA levels, compared with ECs cultured alone. SMCs increased EC NO production when compared with ECs cultured alone. In addition, SMC inhibition of EC ET-1 production could be blocked by the NO synthase inhibitor N(G)-nitro-L-arginine-methyl ester. ECs stimulated SMC proliferation, and the ET-1 AB and B receptor blockers inhibited EC stimulation of SMC proliferation. The ET-1 A blocker had no effect on SMC proliferation. We conclude that SMCs regulate EC ET-1 and ecNOS synthase transcript levels and protein levels. SMC inhibition of ET-1 production by ECs may be mediated through SMC-modulated changes in EC NO activity. Finally, EC stimulation of SMC proliferation in bilayer co-culture is mediated by ET-1 through the ET-1 B receptor.

Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper.

We have developed a method to clone genomic DNA selectively into a yeast-bacterial shuttle vector, pClasper, by recombinogenic targeting in yeast. A gene-specific pClasper targeting vector was constructed with small recombinogenic ends (500 bp) derived from flanking sequences of the genomic region to be cloned. Linearized, recombinogenic pClasper targeting vector and native genomic DNA were cotransformed into yeast. The gene of interest is selectively cloned by recombination between the recombinogenic ends in the targeting vector and homologous regions in the genomic DNA. Here we demonstrate direct cloning of a stably integrated Hoxc8-LacZ-Ura3 reporter gene construct from a mouse embryo fibroblast cell line and single-copy genes from total human genomic DNA. The frequency of capture of the recombinant insert was 0.05-3% of transformants. In contrast to previous reports, we were able to clone genomic DNA directly with a vector containing yeast autonomous replicating sequences. This approach provides a powerful method with which to clone and modify genes precisely for functional analysis.

pPAC-ResQ: A yeast-bacterial shuttle vector for capturing inserts from P1 and PAC clones by recombinogenic targeted cloning.

We have developed a method to capture inserts from P1 and P1 artificial chromosome (PAC) clones into a yeast-bacteria shuttle vector by using recombinogenic targeting. We have engineered a vector, pPAC-ResQ, a derivative of pClasper, which was previously used to capture inserts from yeast artificial chromosome clones. pPAC-ResQ contains DNA fragments flanking the inserts in P1 and PAC vectors as recombinogenic ends. When linearized pPAC-ResQ vector and P1 or PAC DNA are cotransformed into yeast, recombination between the two leads to the transfer of inserts into pPAC-ResQ. pPAC-ResQ clones thus obtained can be further modified in yeast for functional analysis and shuttled to Escherichia coli to produce large quantities of cloned DNA. This approach provides a rapid method to modify P1/PAC clones for functional analysis.

Recombinogenic targeting: a new approach to genomic analysis--a review.

Currently, recombinational cloning procedures based upon methods developed for yeast, Saccharomyces cerevisiae, are being exploited for targeted cloning and in-vivo modification of genomic clones. In this review, we will discuss the development of large-insert vectors, homologous recombination-based techniques for cloning and modification, and their application towards functional analysis of genes using transgenic mouse model systems.

Overexpression of protein kinase C alpha and beta1 has distinct effects on bovine aortic endothelial cell growth.

Protein kinase C (PKC) plays an important role in the mitogenic response of endothelial cells to growth factors. PKC alpha and beta1 are the predominant classical isoforms expressed by bovine aortic endothelial cells (BAECs). The present studies were undertaken to elucidate the effect of PKC alpha and beta1 overexpression in BAEC growth. A series of BAEC lines that stably overexpress the full-length PKC alpha and beta1 cDNA were generated by using a replication-defective recombinant retrovirus. The level of PKC alpha and beta1 cDNA expression was determined by assaying for PKC alpha and beta1 mRNA transcripts. PKC alpha and beta1 protein levels were analysed by Western blotting. Functional analysis of these overexpressing lines was performed by measuring PKC activity and phorbol ester-binding assays. PKC alpha and beta1 overexpression had distinctive effects on BAEC growth and cell-cycle progression. Relative to untransfected BAECs and BAECs transfected with the viral vector alone, BAECs that overproduced PKC alpha exhibited reduced proliferation in vitro and increased accumulation of cells in the G2/M phase of the cell cycle. Growth inhibition was greater in cell lines overexpressing higher levels of PKC alpha. Conversely, a 5-fold greater increase in PKC beta1 activity promoted BAEC growth and shortened BAEC doubling time, whereas cells with a 2- to 4-fold increase in enzyme activity had growth profiles similar to those of both control groups. These results suggest that PKC alpha and beta1 overexpression has reciprocal effects on BAEC growth.

Endothelial cells inhibit smooth muscle cell secretion of hyaluronanic acid.

OBJECTIVE: Hyaluronanic acid is a glycosaminoglycan that is present soon after arterial injury and that may augment the smooth muscle cell (SMC) response to injury. The effect of bovine endothelial cells (ECs) on bovine SMC hyaluronanic-acid synthesis was assessed with a bilayer coculture model. METHODS: Hyaluronanic acid was measured in conditioned media by means of radioimmunoassay and in the cell matrix by histochemistry and point hit quantitation. The receptor for hyaluronic acid-mediated motility (RHAMM) expression, which is increased by hyaluronanic acid, was measured by means of immunoblot. RESULTS: Hyaluronanic acid levels in the conditioned media of SMCs cultured alone (425+/-30 microg/ml/10(6) cells) were greater at 48 hours when compared with SMCs cocultured with ECs (212+/-30 microg/ml/10(6) cells; p < 0.01). Histochemical stain for hyaluronanic acid showed increased hyaluronanic acid in SMCs cultured alone at all time points studied. This finding was confirmed by point hit quantitation at all time points (p < 0.05). Cocultured SMCs had a 50%+/-17% reduction in RHAMM as compared with SMCs cultured alone (p=0.08). CONCLUSIONS: ECs inhibit SMC hyaluronanic acid synthesis and RHAMM expression. This inhibition may be an important mechanism by which ECs modify the SMC response to injury.

Coculture conditions alter endothelial modulation of TGF-beta 1 activation and smooth muscle growth morphology.

We examined whether endothelial cells (ECs) inhibit smooth muscle cell (SMC) transforming growth factor-beta 1 (TGF-beta 1) activation in bilayer coculture. Western analysis showed that SMCs cocultured with ECs as a bilayer had lower amounts of active TGF-beta 1 protein compared with SMCs cultured alone and SMCs cocultured with ECs as a monolayer. EC inhibition of TGF-beta 1 activation could be blocked with plasminogen activator inhibitor-1 (PAI-1) antibody. Similarly, SMC hill-and-valley growth, a marker for TGF-beta 1 activity, was present in SMCs cultured alone and SMCs cocultured with ECs as a monolayer but was absent in SMCs cocultured as a bilayer. SMCs cocultured with ECs as a bilayer migrated at a greater rate than SMCs cultured either alone or cocultured as a monolayer. The EC effect on SMC migration was inhibited by the addition of 5 ng/ml TGF-beta 1. ECs had no effect on SMC RNA levels of TGF-beta 1. PAI-1 levels were increased in ECs and ECs cocultured with SMCs compared with SMCs cultured alone. ECs inhibit TGF-beta 1 activation in bilayer coculture. This appears to be mediated through an increase in EC PAI-1 release. Alterations in coculture conditions, in particular the degree of EC-SMC cell contact, have profound effects on this process.

Endothelial cell effect on smooth muscle cell collagen synthesis.

UNLABELLED: Extracellular matrix (ECM) constitutes the bulk of mature intimal hyperplastic lesions. To determine if endothelial cells (ECs) inhibit smooth muscle cell (SMC) collagen synthesis, bovine aortic SMCs were cultured on plastic semipermeable membranes either alone or opposite ECs. SMCs were pulsed with 4 microCi/ml [3H]proline for 3 days and collagen synthesis was measured as trichloroacetic acid-precipitable counts in conditioned media and in cell lysate fractions. Each was standardized to SMC DNA (cpm/microgram DNA). EC effect on SMC gene expression of type I collagen was studied using Northern analysis. To determine whether TGF-beta 1 activation is involved in collagen synthesis regulation, parallel studies were performed in the presence of a neutralizing antibody to TGF-beta 1 (25 micrograms/ml) or aprotinin (a plasmin inhibitor, 200 micrograms/ml). SMCs cocultured with ECs were either untreated or treated with 5 ng/ml TGF-beta 1. The effect of culture substrate on EC-SMC interaction was studied by repeating experiments on type I collagen (CI), matrigel (MG), fibronectin (FN), and type IV collagen (CIV). RESULTS: Compared to SMCs cultured alone, ECs inhibited SMC collagen synthesis by 40 +/- 5% (P < 0.01) and gene expression of type I collagen by 60 +/- 4% (P < 0.01). The addition of neutralizing TGF-beta 1 Ab or aprotinin to SMCs cultured alone had no effect on collagen synthesis. Collagen synthesis in SMCs cultured alone on MG was reduced by 49% and by 16% on CIV when compared to SMCs cultured on plastic (plastic: 100 +/- 12% vs MG: 51 +/- 15% vs CIV: 84 +/- 18%, P = 0.10). Collagen synthesis was increased in SMCs cultured alone on FN (195 +/- 14% vs plastic: 100 +/- 12, P < 0.001) and CI (207 +/- 17 vs plastic: 100 +/- 12, P < 0.001). ECs decreased SMC collagen synthesis by 18 +/- 4% when cocultured on CI (P < 0.05) and by 22 +/- 2% when cocultured on MG (P < 0.05). FN prevented EC inhibition of SMC collagen synthesis. CONCLUSIONS: ECs inhibit SMC collagen synthesis and downregulate SMC type I collagen gene expression. TGF-beta 1 does not appear to be involved in this process. Culture substrate composition can affect SMC collagen synthesis and EC modulation of this process.

Pedicled omental transfer for ischaemic limbs--a 5-year experience.

Chronic occlusive arterial diseases form a single largest entity amongst the peripheral vascular diseases. Current operative methods available for improving circulation often elicit poor results and the patient has to undergo an amputation. The technique of pedicled omental transfer has given hope of saving such unsalvageable limbs. Although symptomatic and clinical improvement has been reported by this method of "biological by-pass revascularisation", there are no simple, objective and easily reproducible tests to assess improvement in circulation. In this study pulse oximetry and stress testing have been used to assess revascularisation. This study comprised 56 patients (78 limbs) suffering from chronic occlusive arterial disease, spanning a period of 5 years. Patients were investigated and subjected to pedicled omental transplantation (omentopexy). Symptomatological assessment showed improvement in intermittent claudication in about 85% of patients, relief from rest pain in 86% and healing of chronic ulcers in 73% of patients. Objective tests of stress testing and pulse oximetry also showed improvement in circulation. Relief from ischaemia was more in cases of Buerger's disease (TAO) than in cases of atherosclerosis obliterans (ASO).

Civilian vascular trauma: an experience of 54 cases.

Fifty-four patients with civilian vascular injuries of the extremities caused by blunt trauma in 41 patients and by penetrating trauma in 13 patients were evaluated. Twenty-nine patients (53.7%) had associated fractures/dislocations and 19 sustained concomitant venous injuries. Twenty-nine patients (53.7%) came with a lagging period of more than 12 hours. Vascular injuries were diagnosed both clinically and by Doppler examination. In patients with equivocal findings, arteriography was performed. Arterial repair was done in 19 patients and saphenous vein interposition grafting was done in 2 patients. Fifteen patients required fasciotomy. Amputation was done in 13 patients (24.07%). It is concluded that the time lag, incorrect and incomplete assessment, and the reluctance to perform fasciotomy early and completely are some of the factors responsible for poor results.

Heteroduplex DNA formation and homolog pairing in yeast meiotic mutants.

Previous studies of Saccharomyces cerevisiae have identified several meiosis-specific genes whose products are required for wild-type levels of meiotic recombination and for normal synaptonemal complex (SC) formation. Several of these mutants were examined in a physical assay designed to detect heteroduplex DNA (hDNA) intermediates in meiotic recombination. hDNA was not detected in the rec102, mei4 and hop1 mutants; it was observed at reduced levels in red1, mek1 and mer1 strains and at greater than the wild-type level in zip1. These results indicate that the REC102, MEI4, HOP1, RED1, MEK1 and MER1 gene products act before hDNA formation in the meiotic recombination pathway, whereas ZIP1 acts later. The same mutants assayed for hDNA formation were monitored for meiotic chromosome pairing by in situ hybridization of chromosome-specific DNA probes to spread meiotic nuclei. Homolog pairing occurs at wild-type levels in the zip1 and mek1 mutants, but is substantially reduced in mei4, rec102, hop1, red1 and mer1 strains. Even mutants that fail to recombine or to make any SC or SC precursors undergo a significant amount of meiotic chromosome pairing. The in situ hybridization procedure revealed defects in meiotic chromatin condensation in mer1, red1 and hop1 strains.

Sigmoido-rectal intussusception.

Sigmoido-rectal intussusception is the least common type of intussusception seen in infants and children and is therefore usually misdiagnosed as rectal prolapse. Delay in diagnosis and treatment is due to lack of its awareness amongst surgeons, incomplete assessment of the prolapsed bowel at the anal orifice, and absence of classical traid of intussusception i.e. palpable abdominal mass, colicky abdominal pain, and bleeding per rectum.

Serum lipid peroxide and other enzyme levels of patients suffering from thermal injury.

The levels of the lipid peroxidation product malondialdehyde (MDA) and the activity of transaminases and alkaline phosphatases were estimated in the sera of 25 thermally injured patients at various time intervals after injury. The level of MDA was increased during the early postburn period, whereas the activities of transaminases and alkaline phosphatase became elevated later after injury. It is concluded that an increased concentration of lipid peroxidation product (MDA) in the early postburn period may affect the spleen, liver and kidney, resulting in the release of enzymes into blood stream. Such damage may be checked by the antioxidants superoxide dismutase or allopurinol.

The rec102 mutant of yeast is defective in meiotic recombination and chromosome synapsis.

A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11.