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Biomedical subjects

S Rees

Publications and source records attributed to S Rees.

At least 19 recordsLinked to original sources
PubMed2006

In-patient detoxification procedures, treatment retention, and post-treatment opiate use: comparison of lofexidine + naloxone, lofexidine + placebo, and methadone.

OBJECTIVE: In-treatment and post-treatment outcomes were compared for three detoxification procedures (lofexidine+naloxone, lofexidine+placebo naloxone, and methadone). SAMPLE AND DESIGN: The sample was 137 opiate dependent in-patients. Detoxification treatments were 6-day lofexidine+naloxone (n=45), lofexidine+placebo naloxone (n=46), or 10-day methadone reduction (n=46). A cohort study design was used with double-blind random allocation to lofexidine+naloxone versus lofexidine+placebo. Patients who did not consent to, or who were excluded from randomisation received methadone. RESULTS: Outcome differences between treatment groups at follow-up were generally associated with length of stay post-detoxification rather than detoxification procedure. Among patients who were not opiate abstinent throughout follow-up (n=85), those who received lofexidine+naloxone detoxification reported a longer interval to first heroin use, with an interaction between detoxification medication and subsequent retention in treatment also identified. CONCLUSIONS: Detoxification medication may influence medium-term opiate use outcomes via its effect upon retention in treatment.

Adolescent↗
PubMed2006

Does cognitive function influence alaryngeal speech rehabilitation?

BACKGROUND: We sought to define the cognitive domains that influence valved speech rehabilitation. METHODS: Sixteen laryngectomees with primary tracheoesophageal punctures were randomly recruited from one head and neck unit. They were assessed by a consultant neuropsychologist and a speech therapist. Speech therapy time was determined from speech therapy notes. RESULTS: The Digit Symbol Substitution Test, assessing learning speed and processing speed, correlated significantly with speech therapy time in the first (p = .002) and third (p = .014) postoperative years, respectively. Categorical fluency assessment correlated positively with speech therapy time in the first year (p = .009). Learning speed (p = .007) and categorical fluency (p = .041) correlated positively with the fall in speech therapy input between the first and third year after laryngectomy. CONCLUSIONS: Learning speed, processing speed, and categorical fluency strongly influence alaryngeal speech rehabilitation. This study highlights the potential for pre-laryngectomy cognitive assessment to help plan alaryngeal speech rehabilitation. This has significant resource implications.

Aged↗
PubMed2006

Activation of NF-kappaB transcription factor in the preterm ovine brain and placenta after acute LPS exposure.

Intrauterine infection may be causally related to inflammation and injury of the fetal brain, however the mechanisms by which this occurs are unclear. We have investigated whether nuclear factor (NF)-kappaB, a transcription factor for proinflammatory cytokines, is activated in the fetal brain after acute LPS-exposure. At 95 days of gestation (term = approximately 147 days), 5 fetuses received a single intravenous bolus dose of LPS (1 microg/kg); 6 fetuses served as controls. Fetal blood samples were taken hourly for 6 hr post LPS-exposure to assess physiological status. Ewes and fetuses were then euthanased, placental and brain tissue examined histologically, and NF-kappaB activation assessed in several regions of the fetal brain using an electromobility shift assay (EMSA). Oxidative stress was measured using lipid peroxidation and 8-isoprostane biochemical assays and brain cytokine concentrations analysed by enzyme linked immunosorbent assay (ELISA). LPS-exposed fetuses (relative to controls) were hypoxemic and the haematocrit and lactate levels had increased. In the brains of LPS-exposed fetuses compared to controls, NF-kappaB binding activity was elevated in the hippocampus and the thalamus/basal ganglia; 8-isoprostane levels were elevated overall (P < 0.05) in the parietal/occipital/temporal lobes and thalamus/basal ganglia. TNF-alpha and IL-6 concentrations were not elevated, however, there was a tendency for an elevation of IFN-gamma concentrations in the thalamus/basal ganglia. IFN-gamma concentration was elevated (P < 0.05) in the plasma 4 hr after LPS-exposure. In the placenta, NF-kappaB binding activity was increased (P < 0.05). We conclude that acute systemic administration of LPS leads to increased binding activity of NF-kappaB subunits in specific regions of the fetal brain and in the placenta, but that there is no clear-cut relationship between this elevation and vulnerability to endotoxic damage.

Animals↗
PubMed2004

An animal model of chronic placental insufficiency: relevance to neurodevelopmental disorders including schizophrenia.

Evidence now suggests that compromised prenatal brain development may increase the risk for the manifestation of neurological disorders such as schizophrenia. We present a guinea-pig model which mimics a condition of human pregnancy, namely, chronic placental insufficiency. Previously we reported that at term there are changes in the brains of these offspring which are relevant to changes in patients with schizophrenia. The aim of this study was to examine whether deficits in brain structure persist to adolescence and young adulthood (8-12 weeks) and have implications for behavioral function. Reduced uteroplacental blood flow was induced via unilateral ligation of the uterine artery at mid-gestation. The brain was examined in control and prenatally compromised (PC) animals 8 weeks after birth using morphometric and immunohistochemical markers. In a separate cohort of animals, prepulse inhibition (PPI) of the acoustic startle response was assessed at 4, 8 and 12 weeks of age. Brain neurochemistry was examined by determining the concentrations of dopamine and its metabolite, dihydroxyphenylacetic acid (DOPAC), at 12 weeks using high performance liquid chromatography. In PC animals compared with controls there was a reduction in brain weight, persistent enlargement of the lateral ventricles, a reduction in the volume of the basal ganglia and septal region and no evidence of gliosis. No differences were observed in concentration of catecholamines in any brain region examined. At 12, but not 4 or 8, weeks of age, PPI was reduced in PC animals compared with controls. The findings of reduced brain weight, ventriculomegaly, reduced basal ganglia volume and absence of astrogliosis in the PC guinea-pig brain at adolescence parallel some of the changes observed in patients with schizophrenia. The impairment of PPI is comparable to sensorimotor gating deficits observed in patients with schizophrenia. These results indicate that adverse prenatal conditions lead to long-term alterations in brain structure and function which resemble alterations seen in patients with schizophrenia and therefore support the early neurodevelopmental hypothesis of schizophrenia.

Animals↗
PubMed2003

The effects of repeated endotoxin exposure on placental structure in sheep.

Intrauterine infection has been associated with fetal brain injury and preterm birth. We have recently shown that repeated exposure to bacterial endotoxin leads to hypoxia and brain injury in the preterm ovine fetus and we considered it possible that endotoxin could also damage the placenta. Our aim therefore was to assess placental structure following repeated exposure to endotoxin. Endotoxin was administered on 3-5 occasions (1 microg/kg, i.v.) over 5 days from 95-99 days of gestation (term approximately 147 days) to 6 fetal sheep and placental structure assessed at 105 days. In LPS-exposed animals there was a 17 per cent reduction (P<0.05) in placental weight and the average cross-sectional area of placentomes was reduced (P<0.05) by 20 per cent. In addition, all LPS-exposed placentae showed significant injury as evidenced by calcium deposits associated with areas of infarcted tissue. We conclude that repeated endotoxin exposure results in damage to the placenta which could lead to persistent alterations in placental exchange function.

Animals↗
PubMed2001

Splints/orthoses in the treatment of rheumatoid arthritis.

BACKGROUND: Splints/orthoses are often recommended to patients with rheumatoid arthritis (RA) to decrease pain, decrease swelling and/or prevent deformity. These orthoses include resting hand splints, wrist supports, finger splints and special shoes and shoe inserts. OBJECTIVES: To assess the effectiveness of splints/orthoses in relieving pain, decreasing swelling and/or preventing deformity and determine the impact of splints/orthoses on strength, mobility and function in people with RA. SEARCH STRATEGY: We searched MEDLINE, EMBASE, the PEDro data base and Current Contents up to January 2002, and the Cochrane Controlled Trials Register to Issue 4, 2001 using the search strategy developed by the Cochrane Collaboration. Unpublished studies were sought by hand searching conference proceeding and contacting key experts. SELECTION CRITERIA: All randomized control trials (RCTs) and controlled clinical trials (CCTs), case-control and cohort studies comparing the use of specific orthoses against placebo, another active intervention (including another type of orthoses) or regular treatment were selected, according to an a priori protocol. DATA COLLECTION AND ANALYSIS: Two reviewers independently selected the studies and abstracted data. The methodological quality of the RCTs and CCTs was assessed using a validated scale. MAIN RESULTS: Twelve papers reporting on 10 studies met the inclusion criteria. These studies dealt with the following: working wrist splints (5), resting hand and wrist splints (2), special shoes and insoles (3). There is evidence that wearing working wrist splints statistically significantly decreases grip strength and does not affect pain, morning stiffness, pinch grip, quality of life after up to 6 months of regular wear. We found no evidence that resting wrist and hand splints change pain, grip strength, Ritchie Index or number of swollen joints. However, patients who wore these splints for 2 months reported that they preferred use to non-use and padded resting splints to unpadded ones. The one study of special shoes provided evidence of significant benefits of wearing extra-depth shoes for 2 months including less pain on walking and stair climbing and more minutes pain free walking time. Extra-depth shoes with semi-rigid insoles provided better pain relief than extra-depth shoes alone when worn over 12 weeks. Posted insoles prevented progression of hallux valgus angle but did not affect pain or function. REVIEWER'S CONCLUSIONS: There is insufficient evidence to make firm conclusions about the effectiveness of working wrist splints in decreasing pain or increasing function for people with RA. Potential adverse effects such as decreased range of motion do not seem to be an issue, although some of these splints decrease grip strength and dexterity. Similarly, preliminary evidence suggests that resting hand and wrist splints do not seem to affect range of motion or pain, although patients preferred wearing a resting splint to not wearing one. There is evidence that extra-depth shoes and molded insoles decreases pain on weight-bearing activities such as standing, walking and stair-climbing. Posted insoles may be effective in preventing progression of hallux abductus angle but do not appear to have an impact on pain.

Arthritis, Rheumatoid↗
PubMed2001

The generation and characterisation of antagonist RNA aptamers to MCP-1.

Monocyte chemoattractant protein-1 (MCP-1) has been implicated as a powerful pro-inflammatory mediator and may represent a potentially important, therapeutic opportunity for treatment of inflammatory disease and atherosclerosis. To further investigate the role of MCP-1 in inflammatory disorders we have isolated a series of RNA aptamers that bind specifically to mouse MCP-1. The highest affinity aptamers, designated ADR7 and ADR22, have been functionally characterised in vitro and in cell based assays. ADR7 and ADR22 have an affinity of 180 pM and 370 pM respectively for mouse MCP-1, they can antagonise MCP-1 binding to heparin and specifically antagonise MCP-1 induced chemotaxis in a cell based assay. An interesting feature of ADR22 but not ADR7 is that it is capable of antagonising the function of human MCP-1, demonstrating the high level of specificity of these aptamers and that the aptamers recognise MCP-1 in different ways. The aptamers may be used as a tool to further investigate the role of MCP-1 in inflammatory disorders and may also have a role as a therapeutic agent.

Animals↗
PubMed2001

Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta -opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy.

Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.

Blotting, Western↗
PubMed2001

Reporter-gene systems for the study of G-protein-coupled receptors.

Reporter-gene assays offer an alternative to biochemical assays for following signal transduction pathways from receptors at the cell surface to nuclear gene transcription in living cells. Specific reporter-gene systems are now available for the study of ligand activity at G alpha(i/o), G alpha(s) and G alpha(q) G-protein-coupled receptors. In recent years reporter genes have been applied in academia and industry to the study of ligand efficacy and affinity in recombinant and primary cell lines using a variety of colour, fluorescent or luminescent read-outs.

Animals↗
PubMed2001

Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase-tagged, constitutively active mutant, G-protein-coupled receptor.

The wild-type beta2-adrenoceptor and a constitutively active mutant of this receptor were C-terminally tagged with luciferase from the sea pansy Renilla reniformis. C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant beta2-adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. Treatment of cells expressing constitutively active mutant beta2-adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. The pEC50 for ligand-induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated. Similar upregulation could be observed following sustained treatment with agonist ligands. These effects were only observed at a constitutively active mutant of the beta2-adrenoceptor. Co-expression of the wild-type beta2-adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. Co-expression of the constitutively active mutant beta2-adrenoceptor-Renilla luciferase and an equivalent mutant of the alpha1b-adrenoceptor C-terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. This approach offers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors.

Adenylyl Cyclases↗
PubMed2001

Development of a homogeneous MAP kinase reporter gene screen for the identification of agonists and antagonists at the CXCR1 chemokine receptor.

Agonist activity at G protein-coupled receptors (GPCRs) that regulate heterotrimeric G proteins of the Galpha(i/o) or Galpha(q) families has been shown to result in activation of the mitogen-activated protein (MAP) kinase cascade. To facilitate compound screening for these classes of GPCR, we have developed a reporter gene that detects the activation of the ternary complex transcription factor Sap1a following MAP kinase activation. In contrast to other reporter gene assays for Galpha(i/o)-coupled GPCRs, the MAP kinase reporter generates an increase in signal in the presence of agonist. The reporter gene has been transfected into Chinese hamster ovary cells to generate a "host" reporter gene-containing cell line. The Galpha(i)-coupled human CXCR1 chemokine receptor was subsequently transfected into this cell line in order to develop a 384-well format screen for both agonists and antagonists of this receptor. Agonists activated the reporter gene with the expected rank order of potency and with similar concentration dependence as seen with the regulation of other signal transduction cascades in mammalian cells: interleukin-8 (IL-8) (pEC(50) = 7.0 +/- 0.1) > GCP-2 (pEC(50) = 6.3 +/- 0.1) > NAP-2 (pEC(50) < 6). CXCR1-mediated activation of MAP kinase was inhibited by pertussis toxin and the MEK inhibitor PD98059, demonstrating that receptor activation of MAP kinase is due to pertussis toxin-sensitive Galpha(i/o)-family G proteins to cause the activation of MEK kinase. Using the 384-well format, assay performance was unaffected by solvent concentrations of 0.5% ethanol, 0.15% glycerol, or 1% DMSO. Signal crosstalk between adjacent wells was less than 1%. The assay exhibited a Z factor of 0.53 and a coefficient of variation of response to repeated application of IL-8 (100 nM) of 15.9%.

Animals↗
PubMed2001

Application of beta-galactosidase enzyme complementation technology as a high throughput screening format for antagonists of the epidermal growth factor receptor.

We have applied enzyme complementation technology to develop a screen for antagonists of the epidermal growth factor (EGF) receptor. Chimeric proteins containing two weakly complementing deletion mutants of Escherichia coli beta-galactosidase (beta-gal), each fused to the EGF receptor extracellular and transmembrane domains, have been stably expressed in C2C12 cells. In this cell line, formation of active beta-gal is dependent on agonist-stimulated dimerization of the EGF receptor. We have developed a homogenous 384-well assay protocol and have applied this to characterize the pharmacology of the receptor and to develop a high throughput screen (HTS) for EGF receptor antagonists. The assay is tolerant to DMSO concentrations of up to 2% and, across 21 passages in culture, exhibits an EC(50) for EGF of 5.4 +/- 3.6 ng/ml (n = 11) and a Z' of 0.55 +/- 0.13 (n = 11). A random set of 1,280 compounds was screened in duplicate at 11 microM to examine the robustness of enzyme complementation technology and to characterize the false-positive hit rate in the assay. Using a cutoff of 40% inhibition of EGF-promoted beta-gal activity, the hit rate on day 1 was 2.5% and on day 2 was 1.9%. After retesting the active compounds, the hit rate was reduced to 0.4%, of which one of the compounds was identified as a beta-gal inhibitor and the remainder appeared to be nonspecific inhibitors in the assay. This technology is amenable to automated screen workstations, there are highly sensitive chemiluminescent and fluorescent beta-gal assay reagents amenable to detection in miniaturized plate formats, and the assay benefits from a low false-positive hit rate. Enzyme complementation technology may have wide application within the HTS environment for the detection of modulators of receptor activation or inhibitors of protein-protein interactions in mammalian cells.

Animals↗
PubMed2000

The generation and characterization of antagonist RNA aptamers to human oncostatin M.

Oncostatin M (OSM) is a multifunctional member of the interleukin-6 cytokine family. OSM has been implicated as a powerful proinflammatory mediator and may represent a potentially important, novel therapeutic opportunity for treatment of established rheumatoid arthritis. To further investigate the role of OSM in inflammatory disorders, we have isolated a series of RNA aptamers that bind specifically to human OSM. The highest affinity aptamer, designated ADR58, has been characterized in a series of in vitro and cell based assays. ADR58 has an affinity of 7 nm for human OSM, and it can antagonize OSM binding to the gp130 receptor and specifically antagonize OSM mediated signaling. The aptamer has been truncated in length to 33 bases, all pyrimidine positions are substituted with 2' fluorine, and 14 of 18 purine positions have been substituted with 2' O-methyl to increase stability toward nucleases. This truncated, modified form of ADR58 retains complete affinity and functional activity for OSM. This aptamer may be used as a tool to further investigate the role of OSM in inflammatory disorders and may also have role as a therapeutic agent.

Animals↗
PubMed2000

Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors.

The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.

Animals↗
PubMed2000

Exposure to prenatal carbon monoxide and postnatal hyperthermia: short and long-term effects on neurochemicals and neuroglia in the developing brain.

The effects of prenatal exposure to carbon monoxide (CO), a major component of cigarette smoke, was studied alone or in combination with postnatal hyperthermia, on the structural and neurochemical development of the postnatal brain at 1 and 8 weeks. Pregnant guinea pigs (n = 11) were exposed to 200 p.p.m CO for 10 h/day from midgestation until term (68 days), whereas control mothers (n = 10) breathed room air. On postnatal day 4, neonates from the control and CO-exposed pregnancies were exposed to hyperthermia (35 degrees C) for 75 min or remained at ambient (23 degrees C) temperature. Using semiquantitative immunohistochemical techniques the following neurotransmitter alterations were found in the medulla at 1 week: a decrease in met-enkephalin-immunoreactivity (IR) following postnatal hyperthermia and an increase in 5-hydroxytryptamine-IR following a combination of CO and hyperthermia. No alterations were observed in substance P- or tyrosine-hydroxylase-IR in any paradigm. At 8 weeks of age the combination of prenatal CO exposure followed by a brief hyperthermic stress postnatally resulted in lesions throughout the brain and an increase in glial fibrillary acidic protein-IR in the medulla. Such effects on brain development could be of relevance in cardiorespiratory control in the neonate and could have implications for the etiology of Sudden Infant Death Syndrome, where smoking and hyperthermia are major risk factors.

Animals↗