Search PubMed⌕ Search

Biomedical subjects

John Marshall

Publications and source records attributed to John Marshall.

At least 73 records · Page 4Linked to original sources
PubMed2004

Fluorophore-assisted retinal break detection using antibodies to glial fibrillary acidic protein.

PURPOSE: To evaluate the role of fluorescent antibodies as a means of enhancing the detection of retinal breaks during vitrectomy for rhegmatogenous retinal detachment. METHODS: In ex vivo studies, unfixed, porcine retinal flatmounts were incubated with Cy3 anti-GFAP. Experiments were repeated in the presence of excess soluble GFAP and after surface excimer laser ablation through the internal limiting membrane, into the Müller cell foot processes. Tissue was also incubated with trypan blue, and cross-species immunoreactivity was determined in bovine, rabbit, and human retina. In vivo studies were conducted in a porcine model of rhegmatogenous retinal detachment. Cy3 anti-GFAP was injected into the vitreous cavity of eyes with retinal breaks and then rinsed from the eye. Barrier filters were fitted to the operating microscope to allow intraoperative visualization of tissue stained with Cy3. Excitation endoillumination was provided by a 532-nm diode-pumped laser. RESULTS: In ex vivo studies, retinal flatmounts exposed to Cy3 anti-GFAP showed minimal surface fluorescence, but exposed glial elements at the cut edge of the flatmount stained brightly, as did those exposed by excimer ablation of the Müller cell membrane. Blocking studies confirmed that binding was antigen specific. Trypan blue colocalized to the cut edge of retinal flatmounts. All species showed high levels of immunoreactivity except rabbit. In vivo studies demonstrated selective intraoperative staining of retinal breaks with a high level of specificity. CONCLUSIONS: Intraoperative vital staining of retinal breaks is possible in an animal model of retinal detachment. Ex vivo studies indicate that this occurs because the Cy3 anti-GFAP selectively binds the intermediate filaments of glial cells with damaged or destroyed cell membranes.

Aged↗
PubMed2004

Safety testing of indocyanine green and trypan blue using retinal pigment epithelium and glial cell cultures.

PURPOSE: Indocyanine green (ICG) and trypan blue have been advocated as vital stains for use during macular surgery. The safety of these agents was tested using a cell culture model. METHODS: Human retinal pigment epithelium (RPE) and Müller cell lines were exposed to ICG over a range of concentrations up to 0.5%, and trypan blue up to 0.2%. Cells were exposed to each dye for 5, 15, or 30 minutes, rinsed, and incubated 24 hours. Cell viability was measured using a mitochondrial dehydrogenase-assay and fluorescent live-dead probe. Experiments were repeated using 0.5% and 1% ICG and 0.06% and 0.12% trypan blue, with follow-up at 0, 1, 5, and 15 days. ICG experiments were repeated in the presence of illumination from a xenon light-source channeled through a surgical endolight, and using reduced osmolarity solutions of 0.1%, 0.5%, and 1% (185 vs. 275 mOsM). RESULTS: There was no clear relationship between cell viability and the concentration of the agent or duration of follow-up, except in RPE cells exposed to 1% ICG. These showed a linear (R(2) 0.9952) decline in viability with time, with a significant reduction by day 15 (P = 0.016). RPE cells exposed to ICG and illumination were not significantly different from the negative control, but when illumination was combined with low osmolarity, viability was reduced (P = 0.0016). ICG and illumination reduced Müller cell viability (P < 0.0001 for both 185 and 275 mOsM). Müller cells incubated with 185 mOsM 1% ICG showed a significant reduction in viability (P < 0.0001) not seen with the 185 mOsM 0.5% or 0.1% solutions or in the low-osmolarity RPE groups. CONCLUSIONS: The combination of exposure to 0.5% ICG and the newer endoillumination light-sources can damage cultured Müller cells. Although the preparations of ICG most commonly used clinically did not produce significant damage, relatively small changes in ICG osmolarity and concentration did. This suggests that safety margins are not large. Trypan blue is safe in a cell culture model.

Cell Line↗
PubMed2004

Safety testing of infracyanine green using retinal pigment epithelium and glial cell cultures.

PURPOSE: To undertake safety testing of infracyanine green (IFCG) in a cell culture model. METHODS: Experiments were undertaken in a cell culture model used previously to perform safety testing of indocyanine green (ICG). Human retinal pigment epithelium (RPE) and Müller cells were exposed to IFCG for 5 minutes, over a range of concentrations up to 0.5%. Experiments were repeated, using double-staining with trypan blue. Cell viability was measured at days 1, 5, and 15 using a mitochondrial dehydrogenase assay and a fluorescent live-dead probe containing calcein and ethidium homodimer-1. Viability was measured after exposure to 0.5% IFCG and 5 minutes of illumination with a vitrectomy endolight powered by a xenon light source. RESULTS: RPE viability was not reduced over the range of concentrations and follow-up intervals. RPE cells exposed to IFCG and illumination had reduced viability relative to the negative control (cells exposed to saline), but not relative to those exposed to saline and illumination. Glial cells showed reduced viability at days 1 and 5, but not day 15. Illumination did not further reduce viability. CONCLUSIONS: IFCG has been advocated as a safer macular vital stain than ICG. These results suggest that it is less likely to produce phototoxicity, but despite being nearly iso-osmolar, IFCG also produces damage in cultured glial cells.

Cell Culture Techniques↗
PubMed2004

Compartmental analysis of taurine transport to the outer retina in the bovine eye.

PURPOSE: To assess the relative resistance presented individually by Bruch's membrane-choroid (BC) and the retinal pigment epithelium (RPE) to movement of taurine between the choroidal circulation and the outer retina. To quantify the effect of light-evoked changes in subretinal potassium concentration on the transepithelial transport of taurine across bovine RPE. METHODS: Transport studies were performed in Ussing chambers with intact and RPE-denuded specimens of BC. RPE viability was monitored by recording transepithelial potential (TEP) and transepithelial resistance (TER). Taurine transport with substrate concentrations in the micro- and millimolar range, reflecting physiological taurine concentrations in plasma, retina, and subretinal space was quantified by high-performance liquid chromatography (HPLC) and radiotracer techniques. Taurine transport was also assessed after apical potassium concentration was lowered from 6.0 to 2.2 mM to mimic the effects of light. RESULTS: Transport of taurine across RPE-BC at a 10-mM substrate concentration increased from 32.92 before to 111.72 nanomoles/4 mm per hour after removal of the RPE. Similarly, at 50 microM taurine, transport rates increased from 0.158 to 0.439 nanomoles/4 mm per hour after removal of the RPE. At both high (10 mM) and low (50 microM) substrate concentrations, lowering of apical potassium was associated with decreased transport of taurine across the RPE. For taurine concentrations greater than 42 microM, the rate-limiting compartment for transport of taurine to the outer retina was the RPE monolayer. Similar rates were observed across each compartment for concentrations <42 microM. CONCLUSIONS: The magnitude and directionality of taurine transport across the RPE is determined solely by the driving taurine concentration gradient and is modulated by subretinal levels of potassium. Such modulation may provide a mechanism for conserving retinal taurine. Processes that increase the resistance to diffusion across Bruch's membrane such as human ageing and increased thickening and deposition of debris associated with age-related macular degeneration (AMD) are likely to affect transport across the RPE, culminating in a secondary retinal taurine deficiency.

Animals↗
PubMed2004

Taurine uptake by human retinal pigment epithelium: implications for the transport of small solutes between the choroid and the outer retina.

PURPOSE: To characterize the Michaelis-Menten kinetics of the taurine transporter (TT) in retinal pigment epithelium (RPE) freshly isolated from human donor eyes. To identify the rate limiting compartment in the pathway of taurine delivery from the choroidal blood supply to the outer retina composed by Bruch's-choroid (BC) and the RPE in the human older age group. METHODS: In human donor samples (4 melanoma-affected eyes, and 14 control eyes; age range, 62-93 years), radiochemical techniques were used to determine the RPE taurine accumulation at various exogenous concentrations. The transport capability of human RPE was obtained from a kinetic analysis of the high-affinity carrier over a substrate concentration of 1 to 60 microM taurine. RESULTS: Uptake of taurine into human RPE at a taurine concentration of 1 microM was independent of donor age (P > 0.05) and averaged at 2.83 +/- 0.27 (SEM) pmol/10 minutes per 6-mm trephine. Taurine transport by human RPE was mediated by a high-affinity carrier of K(m) 50 microM and V(max) of 267 pmol/10 minutes per 5-mm disc. CONCLUSIONS: In human donor RPE, uptake of taurine remained viable in the age range 62 to 93 years. Taurine transport rates in the RPE were lower than across the isolated BC complex, and thus the data suggest that the former compartment houses the rate-limiting step in the delivery of taurine to the outer retina.

Aged↗
PubMed2004

TRICOM: enhanced vaccines as anticancer therapy.

Renewed interest in immune therapies for the treatment of cancer has sparked a number of different immune stimulation approaches. These include antibody based, dendritic cell-based and vaccine-based therapies. The poxvirus-based vaccines incorporating a TRIad of COstimulatory Molecules (TRICOM) will be reviewed here. Through stimulation of cancer-specific T-cell responses, researchers have demonstrated interesting clinical efficacy in addition to the high safety profile in clinical trials.

Cancer Vaccines↗
PubMed2003

Cell surface expression of GluR5 kainate receptors is regulated by an endoplasmic reticulum retention signal.

Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.

Alternative Splicing↗
PubMed2003

Withdrawal of mechanical ventilation in anticipation of death in the intensive care unit.

BACKGROUND: In critically ill patients who are receiving mechanical ventilation, the factors associated with physicians' decisions to withdraw ventilation in anticipation of death are unclear. The objective of this study was to examine the clinical determinants that were associated with the withdrawal of mechanical ventilation. METHODS: We studied adults who were receiving mechanical ventilation in 15 intensive care units, recording base-line physiological characteristics, daily Multiple Organ Dysfunction Scores, the patient's decision-making ability, the type of life support administered, the use of do-not-resuscitate orders, the physician's prediction of the patient's status, and the physician's perceptions of the patient's preferences about the use of life support. We examined the relation between these factors and withdrawal of mechanical ventilation, using Cox proportional-hazards regression analysis. RESULTS: Of 851 patients who were receiving mechanical ventilation, 539 (63.3 percent) were successfully weaned, 146 (17.2 percent) died while receiving mechanical ventilation, and 166 (19.5 percent) had mechanical ventilation withdrawn. The need for inotropes or vasopressors was associated with withdrawal of the ventilator (hazard ratio, 1.78; 95 percent confidence interval, 1.20 to 2.66; P=0.004), as were the physician's prediction that the patient's likelihood of survival in the intensive care unit was less than 10 percent (hazard ratio, 3.49; 95 percent confidence interval, 1.39 to 8.79; P=0.002), the physician's prediction that future cognitive function would be severely impaired (hazard ratio, 2.51; 95 percent confidence interval, 1.28 to 4.94; P=0.04), and the physician's perception that the patient did not want life support used (hazard ratio, 4.19; 95 percent confidence interval, 2.57 to 6.81; P<0.001). CONCLUSIONS: Rather than age or the severity of the illness and organ dysfunction, the strongest determinants of the withdrawal of ventilation in critically ill patients were the physician's perception that the patient preferred not to use life support, the physician's predictions of a low likelihood of survival in the intensive care unit and a high likelihood of poor cognitive function, and the use of inotropes or vasopressors.

Adult↗
PubMed2003

Reliability of the visual assessment of cervical and lumbar lordosis: how good are we?

STUDY DESIGN: Blinded test-retest design. OBJECTIVE: To measure the intrarater and interrater reliability of the visual assessment of cervical and lumbar lordosis. SUMMARY OF BACKGROUND DATA: Cervical and lumbar lordoses are frequently evaluated using visual assessment, but little attempt has previously been made to measure the reliability of visual assessment. METHODS: Twenty-eight chiropractors, physical therapists, physiatrists, rheumatologists, and orthopedic surgeons were recruited to evaluate the posture of photographed subjects (with and without back pain). Each clinician rated the lordosis of the cervical and lumbar spines as normal, increased, or decreased. Kappa coefficients (kappa) were calculated to determine intrarater and interrater reliability. RESULTS: Twenty-eight clinicians evaluated photographs of 36 individuals (17 with back pain, 19 without). Mean intrarater reliability was kappa = 0.50 (95% confidence interval 0.02-0.98) and mean interrater reliability was kappa = 0.16 (95% confidence interval 0.00-0.48). No statistically significant difference existed among the five groups of clinicians or between the evaluation of the subjects with and without back pain. CONCLUSION: Intrarater reliability of the visual assessment of cervical and lumbar lordosis was statistically fair, whereas interrater reliability was poor.

Adolescent↗
PubMed2003

Calcium channel and NMDA receptor activities differentially regulate nuclear C/EBPbeta levels to control neuronal survival.

Insulin-like growth factor-1 (IGF-1) promotes the survival of cerebellar granule neurons by enhancing calcium influx through L-type calcium channels, whereas NMDA receptor-mediated calcium influx can lead to excitotoxic death. Here we demonstrate that L and NMDA receptor channel activities differentially regulate the transcription factor C/EBPbeta to control neuronal survival. Specifically, we show that L channel-dependent calcium influx results in increased CaMKIV activity, which acts to decrease nuclear C/EBPbeta levels. Conversely, NMDA receptor-mediated influx rapidly elevates nuclear C/EBPbeta and induces excitotoxic death via activation of the calcium-dependent phosphatase, calcineurin. Moderate levels of AMPA receptor activity stimulate L channels to improve survival, whereas higher levels stimulate NMDA receptors and reduce neuronal survival, suggesting differential synaptic effects. Finally, N-type calcium channel activity reduces survival, potentially by increasing glutamate release. Together, these results show that the L-type calcium channel-dependent survival and NMDA receptor death pathways converge to regulate nuclear C/EBPbeta levels, which appears to be pivotal in these mechanisms.

Animals↗
PubMed2003

Multiple trafficking signals regulate kainate receptor KA2 subunit surface expression.

The kainate receptor subunit KA2 does not form functional homomeric channels despite its structural similarity to the functional glutamate receptor 5-7subunits and high agonist binding affinity in in vitro assays. In this study, we first demonstrate that homomeric KA2 receptors fail to reach the plasma membrane and then identify the molecular mechanisms preventing surface expression. Specifically, we show that KA2 subunits form homooligomeric receptors that are confined to the endoplasmic reticulum (ER). We then demonstrate that, in both heterologous expression systems and primary neurons, the intracellular retention of KA2 is not caused by subunit misfolding but, rather, is mediated through discrete protein trafficking signals, including an arginine-rich ER retention/retrieval motif and a di-leucine endocytic sequence in the C terminus of the KA2 subunit. Disruption of these motifs results in ER exit and surface expression of KA2 homomeric receptors that remain nonfunctional. Furthermore, our data suggest that the ER retention/retrieval signal in KA2 is sterically shielded during heteromeric assembly, allowing delivery of functional heteromeric receptors to the plasma membrane. Taken together, our results illustrate novel regulatory mechanisms that control the intracellular trafficking and surface expression of kainate receptors.

Amino Acid Motifs↗
PubMed2003

Perceptions of patients and physicians regarding phase I cancer clinical trials: implications for physician-patient communication.

PURPOSE: To describe and compare the perceptions of cancer patients and their physicians regarding phase I clinical trials. METHODS: Eligible patients had been offered phase I trial participation and had decided to participate but had not yet begun treatment. Each patient's physician also served as a study subject. Patients and physicians completed questionnaires with domains including perceptions of potential benefit and harm from treatment (experimental and standard), relative value of quality and length of life, and perceived content of patient-physician consultations. RESULTS: Three hundred twenty-eight patients and 48 physicians completed surveys. Patients had high expectations regarding treatment outcomes (eg, median 60% benefit from experimental therapy), with those choosing to participate in a phase I trial being more optimistic than those declining phase I participation. Patients predicted a higher likelihood of both benefit and adverse reactions from treatment (experimental and standard) than their physicians (P <.0001 for all comparisons). Although 95% of patients reported that quality of life was at least as important as length of life, only 28% reported that changes in quality of life with treatment were discussed with their physicians. In contrast, 73% of physicians reported that this topic was discussed (P <.0001). CONCLUSION: Cancer patients offered phase I trial participation have expectations for treatment benefit that exceed those of their physicians. The discordant perceptions of patients and physicians may possibly be explained by patient optimism and confidence; however, the discrepancies in reports of consultation content, particularly given patients' stated values regarding quality of life, raise the possibility that communication in this context is suboptimal.

Adult↗
PubMed2003

Cdk5-mediated inhibition of the protective effects of transcription factor MEF2 in neurotoxicity-induced apoptosis.

Neurotoxic insults deregulate Cdk5 activity, which leads to neuronal apoptosis and may contribute to neurodegeneration. The biological activity of Cdk5 has been ascribed to its phosphorylation of cytoplasmic substrates. However, its roles in the nucleus remain unknown. Here we investigate the mechanism by which Cdk5 promotes neuronal apoptosis. We have identified the prosurvival transcription factor MEF2 as a direct nuclear target of Cdk5. Cdk5 phosphorylates MEF2 at a distinct serine in its transactivation domain to inhibit MEF2 activity. Neurotoxicity enhances nuclear Cdk5 activity, leading to Cdk5-dependent phosphorylation and inhibition of MEF2 function in neurons. MEF2 mutants resistant to Cdk5 phosphorylation restore MEF2 activity and protect primary neurons from Cdk5 and neurotoxin-induced apoptosis. Our studies reveal a nuclear pathway by which neurotoxin/Cdk5 induces neuronal apoptosis through inhibiting prosurvival nuclear machinery.

Amino Acid Sequence↗
PubMed2003

New tendencies and strategies in international immunisation: GAVI and The Vaccine Fund.

It is now accepted that investment in health is an important contributor to poverty reduction; however failure to immunise the world's children with readily available life-saving vaccines results in more than 3 million premature deaths annually. The Vaccine Fund-a new structural tool with a clear mission "every child every where" and the Global Alliance for Vaccines and Immunization (GAVI)-a true alliance of all global stakeholders, are working together to build sustainability of immunisation and immunisation capacity in the world's poorest countries.Substantial funding for immunisation has been raised but more is needed to complete the task of fully immunising the world's poorest children.

Costs and Cost Analysis↗
PubMed2003

Allosteric regulation and spatial distribution of kainate receptors bound to ancillary proteins.

A diverse range of accessory proteins regulates the behaviour of most ligand- and voltage-gated ion channels. For glutamate receptor 6 (GluR6) kainate receptors, two unrelated proteins, concanavalin-A (Con-A) and postsynaptic density protein 95 (PSD-95), bind to extra- and intracellular domains, respectively, but are reported to exert similar effects on GluR6 desensitization behaviour. We have tested the hypothesis that distinct allosteric binding sites control GluR6 receptors via a common transduction pathway. Rapid agonist application to excised patches revealed that neither Con-A nor PSD-95 affect the onset of desensitization. The rate of desensitization elicited by 10 mM L-glutamate was similar in control (taufast = 5.5 +/- 0.4 ms), Con-A-treated patches (taufast = 6.1 +/- 0.5 ms) and patches containing PSD-95 and GluR6 receptors (taufast = 4.7 +/- 0.6 ms). Likewise, the time course of recovery from GluR6 desensitization was similar in both control and Con-A conditions, whereas PSD-95 accelerated recovery almost twofold. Peak and steady-state (SS) dose-response relationships to glutamate were unchanged by lectin treatment (e.g. control, EC50(SS) = 31 +/- 28 microM vs Con-A, EC50(SS) = 45 +/- 9 microM, n = 6), suggesting that Con-A does not convert non-conducting channels with high agonist affinity into an open conformation. Instead, we demonstrate that the effects of Con-A on macroscopic responses reflect a shift in the relative contribution of different open states of the channel. In contrast, the effect of PSD-95 on recovery behaviour suggests that the association between kainate receptors and cytoskeletal proteins regulates signalling at glutamatergic synapses. Our results show that Con-A and PSD-95 regulate kainate receptors via distinct allosteric mechanisms targeting selective molecular steps in the transduction pathway.

Allosteric Regulation↗
PubMed2003

Day hospital vs. home treatment--a comparison of illness severity and costs.

BACKGROUND: Acute home treatment services, providing short-term intensive input as an alternative to in-patient admission, have been recommended by the Department of Health as part of a spectrum of care. The lack of research evidence for such services is in contrast to acute day hospital care which has been better researched, but not widely adopted. This paper compares the patients treated in a randomised controlled trial (RCT) of day hospital vs. in-patient care with patients treated several years later in the home treatment service which developed from the original acute day hospital. METHOD: In the original RCT, patients were randomised at the point of admission to day hospital or in-patient care. The home treatment sample consisted of a consecutive series of admissions. Severity of illness was assessed at admission using the Comprehensive Psychopathological Rating Scale (CPRS). Both samples were followed up for 12 months to monitor service use and costs. RESULTS: Symptom severity among the home treatment sample (n = 71) was greater than the day hospital sample (n = 94) (mean CPRS score 31.6 vs. 25.5, p < 0.0001). This difference remained significant following adjustment for other socio-demographic and illness variables. Secondary care costs for the home treatment sample (including in-patient, home treatment and out-patient costs) were intermediate between the costs for the day hospital and in-patient samples from the RCT, but the differences were not statistically significant. CONCLUSIONS: Extending the remit of an acute day hospital to provide 24-h care and a choice of treatment location is associated with an increase in the severity of illness treated. The impact on costs is unclear and the total cost of the new service may not be significantly less than in-patient care. The results need to be interpreted with caution because of differences in recruitment methods.

Adolescent↗