Biomedical subjects
P Clarke
Publications and source records attributed to P Clarke.
Cost-effectiveness analysis of intensive blood-glucose control with metformin in overweight patients with type II diabetes (UKPDS No. 51).
AIMS/HYPOTHESIS: To estimate the economic efficiency of intensive blood-glucose control with metformin compared with conventional therapy primarily with diet in overweight patients with Type II (non-insulin-dependent) diabetes mellitus. METHODS: Cost-effectiveness analysis based on patient level data from a randomised clinical controlled trial involving 753 overweight (> 120% ideal body weight) patients with newly diagnosed Type II diabetes conducted in 15 hospital-based clinics in England, Scotland and Northern Ireland as part of the UK Prospective Diabetes Study. Subjects were allocated at random to an intensive blood-glucose control policy with metformin (n = 342) or a conventional policy primarily with diet (n = 411). The analysis was based on the cost of health care resources associated with metformin and conventional therapy and the estimated effectiveness in terms of life expectancy gained from within-trial effects. RESULTS: Intensive blood-glucose control with metformin produced a net saving of 258 Pounds per patient (1997 United Kingdom prices) over the trial period (median duration of 10.7 years) due to lower complication costs, and increased life expectancy by 0.4 years (costs and benefits discounted at 6%). CONCLUSIONS/INTERPRETATION: As metformin is both cost-saving in the United Kingdom and extends life expectancy when used as first line pharmacological therapy in overweight Type II diabetic patients, its use should be attractive to clinicians and health care managers alike.
Efficacy study of video-assisted thoracoscopic surgery pleurodesis for spontaneous pneumothorax.
BACKGROUND: This study aims to assess the efficacy of video-assisted thoracoscopic surgery pleurodesis in the treatment of spontaneous pneumothorax with particular reference to the rate of recurrence after abrasion pleurodesis and postoperative neuralgia. METHODS: One hundred one patients who underwent 109 video-assisted thoracoscopic surgery pleurodesis procedures in the Austin & Repatriation Medical Centre between January 1992 and June 1998 were identified from a computerized database. The follow-up period was from 8 months to 7 years and 1 month (mean, 44.4 months). Patients were telephoned and asked as to whether recurrence occurred, and if so, when it occurred and how it was treated. They were asked to grade their current pain level from 0 to 6. RESULTS: Eighty-two patients were contacted, corresponding to 88 video-assisted thoracoscopic surgery pleurodesis procedures that were followed up (80.7%). There were five recurrences (5.7%). The pain level was rated as 0 in 64 cases (72.7%), 1 in 27 cases (23.9%), 2 in 1 case (1.1%), and 3 in 2 cases (2.3%). CONCLUSIONS: These data suggest that video-assisted thoracoscopic surgery pleurodesis is a valid alternative to thoracotomy with pleurectomy for treatment of spontaneous pneumothorax with an acceptable recurrence rate and minimal amount of postoperative neuralgia.
Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti.
Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by Sinorhizobium meliloti. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named rhbABCDEF, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named rhtA, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the rhbABCDEF operon and the rhtA gene was found to be positively regulated by the product of the eighth gene in the cluster, named rhrA, which has characteristics of an AraC-type transcriptional activator. The six genes in the rhbABCDEF operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of S. meliloti 2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for rhbF or rhtA at a time when the nifH gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.
Mental health services. Called to the bench.
All mental health providers must be part of a benchmarking group by December. A group of 26 inner city trusts have developed key indicators and found them useful in monitoring their progress. Research has been useful in highlighting service improvements and remaining areas of difficulty.
Targeting and therapy of carcinoembryonic antigen-expressing tumors in transgenic mice with an antibody-interleukin 2 fusion protein.
The purpose of this study was to engineer a bivalent single-chain anticarcinoembryonic antigen (CEA) antibody and an interleukin 2 (IL-2) fusion protein derivative for selective tumor targeting of cytokines. The variable domains of a high affinity anti-CEA antibody, T84.66, were used to form a single-gene-encoded antibody [single-chain variable fragment joined to the crystallizable fragment, Fc (scFvFc)]. The fusion protein (scFvFc.IL-2) consisted of mouse IL-2-fused to the COOH-terminal end of the scFvFc. The engineered proteins were assembled as complete molecules and were similar to the intact anti-CEA monoclonal antibody (Mab) in antigen-binding properties. Based on IL-2 content of the fusion protein, its ability to support proliferation of CTLL-2 cells was identical with that of IL-2. Despite a molecular size similar to that of the intact Mab, the blood clearance of the fusion protein was markedly faster than that of the intact Mab or scFvFc. Incubation of radiolabeled scFvFc.IL-2 but not the intact or scFvFc antibodies in mouse serum was accompanied by the appearance of complexes, suggesting that the latter may contribute to the accelerated clearance of the fusion protein. Biodistribution and tumor targeting studies were carried out in CEA-transgenic mice bearing CEA-positive murine tumors as well as the antigen-negative parental tumor. The bivalent anti-CEA scFvFc had tumor localization properties similar to those of the intact Mab. Although fusion of IL-2 to the COOH-terminal end of the bivalent scFvFc altered its pharmacokinetic properties, the fusion antibody was able to target tumors specifically. Maximum uptake of the intact Mab, scFvFc, and scFvFc.IL-2 in CEA-positive tumors was 29.3 +/- 5.0, 19.5 +/- 2.1, and 6.6 +/- 0.9% injected dose/g, respectively. Maximum tumor localization ratios (CEA-positive/CEA-negative tumor) were similar for all three antibody types (4.6-6.0), demonstrating the antigen specificity of the tumor targeting. Significant antigen-specific targeting to CEA-positive normal tissues of transgenic mice was not observed. Although the tumor-targeting properties of the fusion protein were low, the growth of CEA-expressing (P = 0.01) but not antigen-irrelevant (P = 0.22) syngeneic tumor cells was inhibited after treatment of transgenic mice with the anti-CEA-IL-2 antibody. Therapy of CEA-expressing tumors was improved after i.v. administration of the fusion protein (P = 0.0001). These studies indicate that anti-CEA antibody-directed cytokine targeting may offer an effective treatment for CEA-expressing carcinomas. The availability of an immunocompetent CEA transgenic mouse model will also help to determine the immunotherapeutic properties of these fusion proteins.
MEK kinase 1 gene disruption alters cell migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect in NF-kappa B activation.
MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase that, in addition to regulating the c-Jun NH(2)-terminal kinase (JNK) pathway, is involved in the control of cell motility. MEKK1(-/-) mice are defective in eyelid closure, a TGFalpha-directed process involving the migration of epithelial cells. MEKK1 expression in epithelial cells stimulates lamellipodia formation, a process required for cell movement. In addition, mouse embryo fibroblasts derived from MEKK1(-/-) mice are inhibited in their migration relative to MEKK1(+/+) fibroblasts. MEKK1 is required for JNK but not NF-kappaB activation in response to virus infection, microtubule disruption, and stimulation of embryonic stem cells with lysophosphatidic acid. MEKK1 is not required for TNFalpha or IL-1 regulation of JNK or NF-kappaB activation in macrophages or fibroblasts. Thus, MEKK1 senses microtubule integrity, contributes to the regulation of fibroblast and epithelial cell migration, and is required for activation of JNK but not NF-kappaB in response to selected stress stimuli.
Human T lymphocyte genetic modification with naked DNA.
Endowing T lymphocytes with novel functional attributes by genetic modification is under development for a broad range of clinical cellular immunotherapy applications. To circumvent many of the limitations associated with viral vector systems, a plasmid-based electroporation system that reliably generates G418-resistant primary human T lymphocyte clones was developed. TCR alpha/beta+ CD4+CD8-, and CD4-CD8+ T lymphocyte clones can be routinely isolated from OKT3-stimulated peripheral blood mononuclear cells electroporated with linear plasmid DNA in a limiting dilution drug selection format. Fluorescence in situ hybridization (FISH) studies performed on T cell metaphase spreads using a probe specific for plasmid sequence demonstrated a single FISH signal doublet that varied in chromosomal location from clone to clone. Southern blot analysis using a Neo-specific probe verified chromosomal integration of plasmid vector at a single site. Band intensity quantitation of blots developed with a zeta-specific probe capable of annealing to both endogenous TCR-zeta and the introduced chimeric zeta sequence demonstrated that integrated plasmid was present at a single copy number. Expression levels of the CD20-specific chimeric immunoreceptor construct from a CMV immediate/early promoter present in the plasmid vector varied widely from clone to clone but remained stable during ex vivo expansion to cell numbers in excess of 10(10). This T lymphocyte genetic modification strategy is currently being piloted in a FDA-sanctioned adoptive therapy trial for recurrent lymphoma.
Current challenges in cancer screening. Part II. Prostate cancer screening.
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More or less equal? Comparing Australian income-related inequality in self-reported health with other industrialised countries.
OBJECTIVE: To measure the distribution of self-reported health by income in order to compare the level of health inequality in Australia with other industrialized countries. METHOD: Using data from the two National Health Surveys undertaken in 1989-90 and 1995, concentration indexes were calculated to quantify the distribution of self-reported health by equivalent income. The concentration index for Australia was compared with those reported for nine industrialized countries in Europe and North America. RESULTS: The estimated income-related concentration indexes were -0.1172 in 1989-90 and -0.1094 in 1995. CONCLUSION: The level of health inequality is not significantly different from the US or the UK, but significantly greater than seven other European nations. IMPLICATIONS: Australia has significant income related health inequalities and the distribution of health appears to be more unequal than in many other industrialized nations. There is a need to further investigate and quantify those features of the anglophone societies that set them apart from some other industrialized nations.
Reovirus-induced apoptosis is mediated by TRAIL.
Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.
Reovirus-induced apoptosis requires activation of transcription factor NF-kappaB.
Reovirus infection induces apoptosis in cultured cells and in vivo. To identify host cell factors that mediate this response, we investigated whether reovirus infection alters the activation state of the transcription factor nuclear factor kappa B (NF-kappaB). As determined in electrophoretic mobility shift assays, reovirus infection of HeLa cells leads to nuclear translocation of NF-kappaB complexes containing Rel family members p50 and p65. Reovirus-induced activation of NF-kappaB DNA-binding activity correlated with the onset of NF-kappaB-directed transcription in reporter gene assays. Three independent lines of evidence indicate that this functional form of NF-kappaB is required for reovirus-induced apoptosis. First, treatment of reovirus-infected HeLa cells with a proteasome inhibitor prevents NF-kappaB activation following infection and substantially diminishes reovirus-induced apoptosis. Second, transient expression of a dominant-negative form of IkappaB that constitutively represses NF-kappaB activation significantly reduces levels of apoptosis triggered by reovirus infection. Third, mutant cell lines deficient for either the p50 or p65 subunits of NF-kappaB are resistant to reovirus-induced apoptosis compared with cells expressing an intact NF-kappaB signaling pathway. These findings indicate that NF-kappaB plays a significant role in the mechanism by which reovirus induces apoptosis in susceptible host cells.
Demonstration of new sites of expression of the CCK-B/gastrin receptor in pancreatic acinar AR42J cells using immunoelectron microscopy.
The CCK-B/gastrin receptor has been characterised in both normal and tumour tissues. Endocytosis of the CCK-B/gastrin receptor has recently been demonstrated and this has similarly been described for other peptide receptors. In addition, ligand and ligand-receptor translocation to the nucleus has been demonstrated for other peptides. The aim of this study was to identify the sites of expression of the CCK-B/gastrin receptor in the known CCK-B/gastrin receptor bearing pancreatic acinar AR42J cells. The specificity of the CCK-B/gastrin receptor antibody (alpha-CCKBR-Ser antibody) was demonstrated by inhibition ELISA studies, radioligand inhibition studies and immunofluorescence binding studies on AR42J cells. Western blotting and immunogold electron microscopy techniques were used to identify the receptor in AR42J cell preparations. The affinity purified alpha-CCKBR-Ser antibody was shown to be specific for the CCK-B/gastrin receptor. The receptor was expressed on the cell membrane, in the cytoplasm and within the nucleus. Isoforms of the receptor protein identified in extra-nuclear and nuclear extracts ranged in molecular weight from 58 to 66 kDa. We conclude that the CCK-B/gastrin receptor is not only expressed on the cell membrane, but also in the cytoplasm and nucleus of AR42J pancreatic acinar cells.
Screening of sulphonamides in egg using gas chromatography-mass-selective detection and liquid chromatography-mass spectrometry.
A rapid extraction and clean-up procedure for sulphonamide antibiotics in eggs suitable for both GC-MSD and LC-MS end determinations has been developed. The drugs were extracted using acetonitrile, acidified using acetic acid and cleaned-up using cation- and anion-exchange. For determination by GC-MSD, extracts were derivatised with diazomethane followed by pentafluoropropionic acid anhydride. For LC-MS extracts were taken up in water and used directly. The methodology developed was validated at the 100 and 25 microg kg(-1) levels, equivalent to the MRL and one quarter of the MRL. Results for the GC-MS procedure were quantitated against deuterated sulphadiazine with relative recoveries ranging from 54% for sulphachloropyridazine to 135.5% for sulphamethazine. Recoveries for the LC-MS procedure ranged from 33% for sulphaguanidine to 92% for sulphamethazine and sulphadimethoxine.
Bcl-2 regulates a caspase-3/caspase-2 apoptotic cascade in cytosolic extracts.
Apoptosis is accompanied by the activation of a number of apoptotic proteases (caspases) which selectively cleave specific cellular substrates. Caspases themselves are zymogens which are activated by proteolysis. It is widely believed that 'initiator' caspases are recruited to and activated within apoptotic signalling complexes, and then cleave and activate downstream 'effector' caspases. While activation of the effector caspase, caspase-3, has indeed been observed as distal to activation of several different initiator caspases, evidence for a further downstream proteolytic cascade is limited. In particular, there is little evidence that cellular levels of caspase-3 that are activated via one pathway are sufficient to cleave and activate other initiator caspases. To address this issue, the ability of caspase-3, activated upon addition to cytosolic extracts of cytochrome c, to cause cleavage of caspase-2 was investigated. It was demonstrated that cleavage of caspase-2 follows, and is dependent upon, activation of caspase-3. Moreover, the activation of both caspases was inhibited by Bcl-2. Together, these data indicate that Bcl-2 can protect cells from apoptosis by acting at a point downstream from release of mitochondrial cytochrome c, thereby preventing a caspase-3 dependent proteolytic cascade.
Antibodies raised by gastrimmune inhibit the spontaneous metastasis of a human colorectal tumour, AP5LV.
Both precursor forms of gastrin and mature amidated gastrin peptides can enhance proliferation of colorectal tumours and may regulate growth in an autocrine manner. The purpose of this study was to evaluate the effect of neutralization of precursor and amidated gastrin on primary and secondary in vivo growth of a human colorectal tumour. The human colorectal cell line, AP5LV, when injected into the muscle layer of the abdominal wall of severe combined immunodeficient (SCID) mice, grows as a well-vascularized primary tumour and metastasis to the lung. AP5LV expressed the precursor gastrin forms; progastrin and glycine-extended gastrin and gastrin/CCKB receptors, as assessed by immunocytochemistry. Gastrimmune is a gastrin immunogen in which the amino terminus of the gastrin-17 molecule is linked to diphtheria toxoid and induces antibodies which neutralise the amidated and glycine-extended forms of gastrin-17. Rabbit antiserum, raised against Gastrimmune, was administered intravenously into SCID mice bearing AP5LV tumours. Control animals were treated with antiserum raised against diphtheria toxoid only. Antibodies raised against Gastrimmune significantly limited the growth of primary AP5LV tumours, as assessed by median cross-sectional area (controls = 244 mm2; antibody-treated = 179 mm2; P = 0.033). In addition Gastrimmune-induced antiserum limited the growth of lung metastasis as assessed by nodule number (controls = 3.5; antibody-treated = 1.0; P = 0.0001) and nodule cross-sectional as assessed by image analysis (controls = 11.9 mm2; antibody-treated = 3.75 mm2; P = 0.0064). In conclusion in vivo neutralization of gastrin forms, which may potentially be fueling growth by an autocrine pathway, inhibited both primary growth and, to a greater degree, lung metastasis of a human colorectal tumour cell line. Immunization against tumour-associated gastrin forms may provide an effective therapy for advanced colorectal cancer.
Intermittent short courses of cyclosporin (Neoral(R)) for psoriasis unresponsive to topical therapy: a 1-year multicentre, randomized study. The PISCES Study Group.
We performed a 1-year study to determine whether intermittent short courses of the microemulsion formulation of cyclosporin (Neoral) could effectively control plaque psoriasis and whether tapering or abrupt cessation of cyclosporin therapy would influence time to relapse. Four hundred patients with plaque psoriasis were included in this open, multicentre, randomized study. All patients commenced cyclosporin at a dose of 2.5 mg/kg daily. Cyclosporin dosage could be increased to a maximum of 5 mg/kg daily. Treatment was continued until clearance of psoriasis or for a maximum of 12 weeks. Patients were then randomly assigned either to stop cyclosporin abruptly or to have the dose reduced by 1 mg/kg daily each week until cessation. On relapse, patients were given another course of cyclosporin. Patients were followed for at least 1 year, during which they could receive as many treatment courses as necessary. The number of patients who required one, two, three and four treatment courses was 400, 259, 117 and 26, respectively. The median time to relapse after the end of the first treatment period was 109 days in the group of patients randomized to stop cyclosporin abruptly and 113 days in patients randomized to taper off cyclosporin (P = 0.038). More than 30% of patients had not relapsed 6 months after having stopped treatment. After each treatment course, the Kaplan-Meier probability of achieving 75% or more reduction in disease area by day 84 of treatment was 83%, 76%, 73% and 66%, respectively. Mean serum creatinine concentration and blood pressure did not show any clinically significant changes over time. Our results show that intermittent short-course therapy with Neoral, when used in conjunction with topical therapy, is well tolerated and provides effective control of plaque psoriasis for 1 year. Tapering off cyclosporin on treatment cessation induces a slight delay in psoriasis relapse.
Comparison of the immunogenicity and safety of two 17D yellow fever vaccines.
As part of the clinical validation process of a new working seed of a licensed yellow fever vaccine (new working seed PV26, Stamaril; Pasteur Mérieux Connaught, Lyon, France), the immunogenicity and safety of two batches of this vaccine (PM-YF) were compared with those of another commercially available vaccine (Arilvax; Evans Medical-Wellcome, Liverpool, United Kingdom) in 211 healthy adults. While the geometric mean titer values at days 10-14 and day 28 after vaccination were higher in the PM-YF group, the vaccines provided equivalent seroprotection (titers > or = 1/10) one month after a single vaccine dose (100% PM-YF versus 99% W-YF; P = 0.001, by one-sided equivalence test). Both vaccines were safe. There were no serious local or systemic reactions reported, nor any clinically significant hepatic function abnormalities associated with the use of either vaccine. These two 17D yellow fever vaccines from different European vaccine manufacturers were highly immunogenic and safe, and provided equivalent seroprotection.