Diabetic retinopathy screening programmes and reducing ophthalmologists' workload.
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Diabetic Retinopathy↗
Biomedical subjects
G Duncan
Publications and source records attributed to G Duncan.
A tetravalent RGD ligand for integrin-mediated cell adhesion.
Monovalent RGD (arginine-glycine-aspartic acid) peptides or polymers furnished with RGD in random distributions are employed as cell-scaffolds and gene delivery vehicles. However, integrin binding to RGD is dependent on the spatial distribution (clustering) of the ligand and intrinsic integrin affinity via conformational changes (avidity). Here we have designed and expressed a polypeptide consisting of a tetrameric coiled coil and spacer facilitating polyvalent (clustered) display of integrin ligands; the RGD motif was used as proof of principle. Size-exclusion chromatography and circular dichroism showed that the polypeptide self assembled as a tetramer in solution with a defined secondary structure. Cell adhesion to surfaces coated with the polypeptide was up to 3-fold greater than that for (monovalent) RGDS peptide at equivalent concentrations. Moreover, the polypeptide in solution at concentrations >or= 1 microM inhibited cell adhesion to fibronectin-coated surfaces, while RGDS peptide in solution at concentrations up to 500 muM did not. These cell data demonstrate that the polypeptide bound integrin receptors in a polyvalent manner. The polypeptide will therefore be of use in the engineering of tissue-culture scaffolds with increased cell adhesion activity, or to targeted gene delivery vehicles, and could incorporate protein ligands in place of the RGD motif.
Amino Acid Sequence↗
Divergent mechanisms of cis9, trans11-and trans10, cis12-conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach.
Conjugated linoleic acids (CLA) affect atherogenesis, but mechanisms are not well understood. We explored how two isomers of CLA, cis9, trans11-CLA and trans10, cis12-CLA, affected lipid and glucose metabolism, as well as hepatic protein expression, in apolipoprotein E knockout mice. After 12 wk of intervention, plasma triglyceride, NEFA, and glucose concentrations were significantly higher in the trans10, cis12-CLA group, whereas plasma triglyceride, NEFA, glucose, and insulin concentrations were significantly lower in the cis9, trans11-CLA group, compared with control mice consuming linoleic acid. Proteomics identified significant up- or down-regulation of 113 liver cytosolic proteins by either CLA isomer. Principal component analysis revealed that the treatment effect of cis9, trans11-CLA was mainly explained by the up-regulation of different posttranslational forms of heat shock protein 70 kD. In contrast, the treatment effect of trans10, cis12-CLA was mainly explained by up-regulation of key enzymes in the gluconeogenic, beta-oxidation, and ketogenesic pathways. Correlation analysis again emphasized the divergent effects of both CLA isomers on different pathways, but also revealed a linkage between insulin resistance and increased levels of hepatic serotransferrin. Thus, our systems biology approach provided novel insights into the mechanisms by which individual CLA isomers differentially affect pathways related to atherogenesis, such as insulin resistance and inflammation.
Animal Feed↗
Outbreak of Candida rugosa mastitis in a dairy herd after intramammary antibiotic treatment.
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Ampicillin↗
Characterisation of TGF-beta2 signalling and function in a human lens cell line.
There is increasing evidence implicating Transforming growth factor beta (TGF-beta) in pathological states of the lens. However, the underlying signalling mechanisms in human cells have not been fully examined. We have therefore investigated in a human lens cell line, FHL 124, the signalling characteristics of TGF-beta and Smad proteins. Moreover, we have tested the effectiveness of a fully human monoclonal anti-TGF-beta2 antibody, CAT-152, in suppressing TGF-beta2 induced changes in a number of conditions. FHL 124 cells were routinely cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% FCS. Characterisation of the cell line was determined using Affymetrix gene microarrays and compared to native human lens epithelium. Cells were serum starved for 24 hr prior to exposure to TGF-beta2 in the presence and absence of CAT-152. Non-stimulated cells served as controls. Smad 4 localisation was observed by immunocytochemistry. To study Smad-dependent transcriptional activity, cells were transfected with SBE4-luc, an artificial smad-specific reporter, using Fugene-6. Transcriptional activity was determined by luciferase activity. Gene expression was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). Proliferation was determined by 3H-thymidine DNA incorporation. Growth and contraction were assessed using a scratch and patch assay. Affymettrix gene microarrays identified 99.5% homology between FHL 124 cells and the native lens epithelium with respect to expression pattern of the 22,270 genes on the chip. Moreover, FHL 124 cells expressed phenotypic markers, alphaA-crystallin and pax6 along with lens epithelial cell specific marker FoxE3. Immunocytochemical studies revealed the presence of Smad 4 which following TGF-beta2 exposure accumulated in the cell nucleus. Furthermore, Smad-dependent transcriptional activity was also stimulated. TGF-beta2 enhanced the expression of mRNA levels of alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Exposure to TGF-beta2 resulted in a relatively small inhibition of 3H-thymidine incorporation of FHL 124 cells. However, a more marked contractile effect was also observed. In serum-supplemented medium, growth rates and TGF-beta induced contraction were enhanced. Treatment with 0.1-10 microg ml(-1) CAT-152 dose-dependently inhibited 10 ng ml(-1) TGF-beta2 induced effects in the presence and absence of serum. Exposure of FHL 124 cells to TGF-beta therefore induces Smad translocation, transcription, expression of transdifferentiation markers and induces marked contraction. Treatment with CAT-152 can effectively inhibit these responses. TGF-beta2 induced changes can also persist long after the period of exposure and when in the presence of serum TGF-beta induced contraction is enhanced. The work presented therefore demonstrates a platform technology to study TGF-beta2 signalling in human lens epithelial cells and provides evidence to show TGF-beta2 can be a potent factor in the development of posterior capsule opacification following cataract surgery.
Actins↗
Bacteraemia and antibiotic use in respiratory syncytial virus infections.
AIMS: To examine the frequency of and risk factors for bacteraemia in children hospitalised with respiratory syncytial virus (RSV) infection; and to determine current use of antibiotics in hospitalised children with RSV infection. METHODS: Retrospective study of all children, aged 0-14 years, admitted to a tertiary children's hospital with proven RSV infection over a four year period. Children with concurrent bacteraemia and RSV infection were identified, and risk factors examined for bacteraemia. The case notes of a randomly selected comparison sample of 100 of these RSV infected children were examined to assess antibiotic use and population incidence of risk factors for severe RSV infection. RESULTS: A total of 1795 children had proven RSV infection, and blood cultures were sent on 861 (48%). Eleven (0.6%) of the 1795 RSV positive children had bacteraemia. RSV positive children had a significantly higher incidence of bacteraemia if they had nosocomial RSV infection (6.5%), cyanotic congenital heart disease (6.6%), or were admitted to the paediatric intensive care unit (2.9%). Forty five (45%) of the random comparison sample of RSV infected children received antibiotics. CONCLUSIONS: Bacteraemia is rare in RSV infection. Children with RSV infection are more likely to be bacteraemic, however, if they have nosocomial RSV infection, cyanotic congenital heart disease, or require intensive care unit admission.
Adolescent↗
Marek's disease, candidiasis and megabacteriosis in a flock of chickens (Gallus gallus domesticus) and Japanese quail (Coturnix japonica).
An outbreak of mortality in chickens and Japanese quail sharing the same airspace was investigated. Marek's disease was diagnosed in five of 11 chickens examined, and in 20 of 24 quail; crop candidiasis was found in four of the chickens and in five of the quail, and moderate to large numbers of organisms referred to as megabacteria were observed in eight of the chickens and 16 of the quail. The disease was so severe that almost all of the quail in the flock died or were culled during the following six months. In contrast, only approximately 5 per cent of the chickens died from Marek's disease.
Animal Feed↗
Role of the non-neuronal cholinergic system in the eye: a review.
Pharmacologically active preparations directed towards modulating muscarinic receptor activity in the eye have been used for over 2000 years when extracts from Atropa belladonna were first applied to enhance eye appearance through pupillary dilation. The first clinically active drugs targeting a specific eye disease were anticholinesterases (e.g. ecothiophate) applied as eye drops to treat glaucoma in the 1960's. However, cataract was soon detected as a relatively frequent side effect and such drugs are now only used to treat glaucoma as a last resort. As muscarinic agonists have been found to reduce intraocular pressure both by decreasing aqueous humour production (through Na,K-ATPase pump inhibition) and increasing outflow (by muscle contraction), it is likely that treatments will be developed that target specific muscarinic subtypes. Recently, it has been shown that the M1 receptor subtype predominates in the lens. It is therefore important that this subtype is not targeted in future ocular therapies so that the side-effect of cataract is avoided. Form-deprived myopia resulting from an increased axial length in the affected eye can be reduced by the application of atropine. This effect has been achieved both in a chick model system and in human clinical trials, and in the former system atropine has been shown to reduce the production of scleral extracellular proteins. Carbachol stimulates tear fluid production through the activation of muscarinic receptors. Interestingly, at least part of the stimulation occurs via epidermal growth factor (EGF) receptors and although the precise signalling mechanisms are not completely understood, it has been shown that calcium mobilisation plays a critical role in both muscarinic and EGF receptor activity. It should be noted that in the four examples described above, the cell types responsible for producing the physiological output are non-neuronal in origin. Therefore cholinergic receptor activation plays diverse roles in the eye and pharmacological intervention based on specific receptor sub-types has potential benefit in a number of ocular problems. However, potential side effects have also recently been identified.
Acetylcholine↗
Epithelial transdifferentiation and cataract in the human lens.
Anterior subcapsular cataracts cause a serious loss of vision and are normally associated with ocular trauma, inflammation or clinical skin conditions. They appear to be accompanied by epithelial cell growth and transdifferentiation where unscheduled production of a number of proteins, including alpha smooth muscle actin (alpha-sma), occurs. Clinical studies have also revealed an up-regulation of the TGFbeta signalling pathway in such cataracts. The present study, using phase contrast and immunofluorescent techniques, was undertaken to investigate the extent of alpha-sma expression in traumatic cataracts, in capsulorhexis specimens obtained during cataract surgery and in aged human lenses from donor eyes. The donor lenses were also exposed to trauma or TGFbeta in culture to observe their relative contribution to alpha-sma production. Dense anterior subcapsular cataracts were relatively rare (<1%), but all showed a pronounced up-regulation of alpha-sma, which was located both in anterior cells of normal appearance and in nucleated fibroblastic cells lying beneath the anterior epithelium. Surprisingly, more than 50% of capsulorhexis specimens from mature cataracts showed expression of alpha-sma, although to a limited extent. Alpha-sma was not expressed in any of the clear donor lenses and culture for 8 days in EMEM did not induce expression. Interestingly, unlike their young animal counterparts, human lenses failed to show the presence of alpha-sma when exposed to 10 ng ml(-1) TGFbeta. However, after culture, lenses with pre-existing cortical opacities did express alpha-sma, as did clear lenses subjected to injury or trauma. It appears that the greater the stress, the greater is the expression of alpha-sma. Cataract, and especially cortical cataract, should therefore be seen as associated with stress-induced signalling pathways in the lens that lead to the transdifferentiation of the anterior epithelial cells.
Actins↗
Stress-induced ATP release from and growth modulation of human lens and retinal pigment epithelial cells.
ATP release has been shown to occur following stimulation in several cellular systems. This study was undertaken to determine if lens and retinal epithelial cells release ATP in response to physiological stresses and to elucidate a possible role for ATP. Analysis of human aqueous humour samples showed a mean ATP level of 37.8+/-7.7 nM. Hyper-osmotic stress induced a dose- and time-dependent release of ATP. Both cell types were found to proliferate in serum-free medium, and the addition of ATP and adenosine at concentrations as low as 0.1 nM inhibited growth. Gene profiling also demonstrated the presence of the ectonucleotidases CD39 and CD73 and the A1 adenosine receptor on both cell types.
Adenosine Triphosphate↗
Nasal fracture manipulation: a comparative study of general and local anaesthesia techniques.
Nasal injuries are common conditions treated in either Otolaryngology or Plastic Surgical departments. Manipulation for deformity can be conducted in various ways. The aim of this study is to determine if the anaesthetic technique used for manipulation influences outcomes. Five hundred and fifty-five patients had either local anaesthetic (LA) or general anaesthetic (GA) nasal fracture manipulations in our departments over a 6-year period. Three hundred and twenty-four of these could be contacted and questioned as to subsequent surgical treatments received. Rhinoplasty, septorhinoplasty or septoplasty had been subsequently performed in 3.2% of the GA group and in 17.2% of the LA group (P < 0.0001). We recommend considering this result when treating nasal fractures in conjunction with other important issues of patient preference, financial costs, associated risks, morbidity and facilities available.
Adolescent↗
Etiological point mutations in the hereditary multiple exostoses gene EXT1: a functional analysis of heparan sulfate polymerase activity.
Hereditary multiple exostoses (HME), a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The corresponding gene products, exostosin-1 (EXT1) and exostosin-2 (EXT2), are type II transmembrane glycoproteins which form a Golgi-localized heterooligomeric complex that catalyzes the polymerization of heparan sulfate (HS). Although the majority of the etiological mutations in EXT are splice-site, frameshift, or nonsense mutations that result in premature termination, 12 missense mutations have also been identified. Furthermore, two of the reported etiological missense mutations (G339D and R340C) have been previously shown to abrogate HS biosynthesis (McCormick et al. 1998). Here, a functional assay that detects HS expression on the cell surface of an EXT1-deficient cell line was used to test the remaining missense mutant exostosin proteins for their ability to rescue HS biosynthesis in vivo. Our results show that EXT1 mutants bearing six of these missense mutations (D164H, R280G/S, and R340S/H/L) are also defective in HS expression, but surprisingly, four (Q27K, N316S, A486V, and P496L) are phenotypically indistinguishable from wild-type EXT1. Three of these four "active" mutations affect amino acids that are not conserved among vertebrates and invertebrates, whereas all of the HS-biosynthesis null mutations affect only conserved amino acids. Further, substitution or deletion of each of these four residues does not abrogate HS biosynthesis. Taken together, these results indicate that several of the reported etiological mutant EXT forms retain the ability to synthesize and express HS on the cell surface. The corresponding missense mutations may therefore represent rare genetic polymorphisms in the EXT1 gene or may interfere with as yet undefined functions of EXT1 that are involved in HME pathogenesis.
Amino Acid Sequence↗
Prepuce pollicization: a warning.
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Adolescent↗
ICOS is essential for effective T-helper-cell responses.
The outcome of T-cell responses after T-cell encounter with specific antigens is modulated by co-stimulatory signals, which are required for both lymphocyte activation and development of adaptive immunity. ICOS, an inducible co-stimulator with homology to CD28, is expressed on activated, but not resting T cells, and shows T-cell co-stimulatory function in vitro. ICOS binds specifically to its counter-receptor B7RP-1 (refs 5,6,7), but not to B7-1 or B7-2. Here we provide in vivo genetic evidence that ICOS delivers a co-stimulatory signal that is essential both for efficient interaction between T and B cells and for normal antibody responses to T-cell-dependent antigens. To determine the physiological function of ICOS, we generated and characterized gene-targeted ICOS-deficient mice. In vivo, a lack of ICOS results in severely deficient T-cell-dependent B-cell responses. Germinal centre formation is impaired and immunoglobulin class switching, including production of allergy-mediating IgE, is defective. ICOS-deficient T cells primed in in vivo and restimulated in vitro with specific antigen produce only low levels of interleukin-4, but remain fully competent to produce interferon-gamma.
Animals↗
In vitro testing of the bond between soft materials used for maxillofacial prostheses and cast titanium.
STATEMENT OF PROBLEM: The choice of soft materials for maxillofacial prostheses is important for covering extraoral defects after tumor surgery or radiation therapy. The use of cast commercially pure titanium as reinforcement seems to be a suitable option. PURPOSE: Making use of the advantages of titanium frameworks for maxillofacial prostheses requires exploring ways of combining it with soft materials. This study investigated such combinations and evaluated discoloration of the soft material to determine whether it was attributable to the titanium reinforcement. MATERIAL AND METHODS: Five soft materials used for maxillofacial prostheses and their bond strengths to cast titanium were tested. Different ways of conditioning the titanium surface and different adhesives were used. Plain mechanical retention was also studied. The Gretag SPM 100 was used to determine the potential effects on coloring after 24 hours of radiation in the Suntest rapid exposure unit. RESULTS: Suitable material combinations for bonding soft materials to titanium were found for all soft materials studied. Bond strength varied depending on the chemical basis of the soft material. Results indicated that maximal bond strengths were obtained by the combinations Supersoft with Super-Bond, Supersoft with subsequent silicoating, and bonding with Dentacolor connector. Few color shifts attributable to the titanium reinforcement were evident in the 2-mm layers of the soft materials applied. CONCLUSION: The results of this study showed that it is possible to combine a titanium framework and soft silicone materials.
Acrylic Resins↗
Interlenticular opacification in piggyback AcrySof intraocular lenses: explantation technique and laboratory investigations.
BACKGROUND/AIMS: Interlenticular opacification (ILO) is a recognised complication of piggyback intraocular lenses (IOLs). The aetiology, histopathology, and treatment are not clearly defined, however. METHODS: Two pairs of AcrySof IOLs were explanted from a patient with bilateral ILO. The explantation technique and surgical challenges of IOL exchanges are described. The explanted IOL complexes and a sample of the anterior capsule were examined by phase, polarising, and immunofluorescence microscopy. RESULTS: A 50 year old man developed ILO bilaterally after piggyback AcrySof IOL implantation. A central contact zone was surrounded by a homogeneous paracentral opacity possibly consisting of extracellular matrix previously laid down by proliferating lens epithelial cells (LECs). These opacities were in turn surrounded by interlenticular Elschnig pearl-type opacities contiguous with the same material filling the periphery of the capsular bag. The IOL complexes were very adherent to the capsular bag and they had to be separated with the help of high viscosity viscoelastic before a single one piece PMMA IOL implantation via large limbal incisions. The sample of anterior capsule showed a ridge configuration from the piling of LECs in the site of apposition with the anterior capsule and cells showing different characteristics on either side of the ridge. CONCLUSION: Cellular proliferation, deposition of ECM from proliferating LECs, and capsular changes induced by cell metaplasia may lead to ILO formation in piggyback AcrySof IOLs. Careful separation of the AcrySof IOL complex from the capsule, meticulous clean up of the proliferating material, and implantation of single or dual in the bag PMMA IOLs through a large incision with capsulorrhexis enlargement may help in the prevention of recurrence of interface opacification.
Cataract↗
The link between heparan sulfate and hereditary bone disease: finding a function for the EXT family of putative tumor suppressor proteins.
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Acetylglucosamine↗
The disposition of gemifloxacin, a new fluoroquinolone antibiotic, in rats and dogs.
Gemifloxacin is a fluoroquinolone antibacterial compound with enhanced affinity for bacterial topoisomerase IV and is being developed for the treatment of respiratory and urinary tract infections. The disposition and metabolic fate of this antibiotic was studied in the rat and the dog, the animal species used in its toxicological evaluation. The investigations were carried out following oral and intravenous administration of gemifloxacin mesylate. Gemifloxacin is a racemic compound; therefore, the pharmacokinetics of its individual (+) and (-) enantiomers were characterized using a chiral high-performance liquid chromatography/tandem mass spectrometry assay. In both rat and dog, the pharmacokinetic profiles of the (+) and (-) enantiomers were essentially identical. The enantiomers were rapidly absorbed following oral administration of racemic gemifloxacin mesylate. They distributed rapidly beyond total body water, and their blood clearance values were approximately equal to one quarter of the hepatic blood flow in each species. Terminal phase elimination half-lives were ca. 2 h in the rat and 5 h in the dog. Gemifloxacin was metabolized to a limited extent following oral and intravenous administration of [14C]gemifloxacin mesylate, and all metabolites formed were relatively minor. The principal metabolites formed were the E-isomer (4-6% of dose) and the acyl glucuronide of gemifloxacin (2-6% of dose) in both species and N-acetyl gemifloxacin (2-5% of dose) in the rat. Data obtained following intravenous administration indicated that gemifloxacin-related material is eliminated from the body via urinary excretion, biliary secretion, and gastrointestinal secretion. Material was eliminated approximately equally by the three routes in the dog, whereas a slightly higher proportion of the dose was eliminated in the urine (46%) and a lower proportion in the bile (12%) of rats.
Administration, Oral↗