Biomedical subjects

M P Smith

Publications and source records attributed to M P Smith.

At least 19 recordsLinked to original sources

PubMed2006

Oxidative stress and dopamine depletion in an intrastriatal 6-hydroxydopamine model of Parkinson's disease.

Although the etiology of Parkinson's disease (PD) is unknown, a common element of most theories is the involvement of oxidative stress, either as a cause or effect of the disease. There have been relatively few studies that have characterized oxidative stress in animal models of PD. In the present study a 6-hydroxydopamine (6-OHDA) rodent model of PD was used to investigate the in vivo production of oxidative stress after administration of the neurotoxin. 6-OHDA was injected into the striatum of young adult rats and the production of protein carbonyls and 4-hydroxynonenal (HNE) was measured at 1, 3, 7, and 14 days after administration. A significant increase in both markers was found in the striatum 1 day after neurotoxin administration, and this increase declined to basal levels by day 7. There was no significant increase found in the substantia nigra at any of the time points investigated. This same lesion paradigm produced dopamine depletions of 90-95% in the striatum and 63-80% in the substantia nigra by 14-28 days post-6-OHDA. Protein carbonyl and HNE levels were also measured in middle-aged and aged animals 1 day after striatal 6-OHDA. Both protein carbonyl and HNE levels were increased in the striatum of middle-aged and aged animals treated with 6-OHDA, but the increases were not as great as those observed in the young adult animals. Similar to the young animals, there were no increases in either marker in the substantia nigra of the middle-aged and aged animals. There was a trend for an age-dependent increase in basal amounts of oxidative stress markers when comparing the non-lesioned side of the brains of the three age groups. These results support that an early event in the course of dopamine depletion following intrastriatal 6-OHDA administration is the generation of oxidative stress.

Age Factors↗

PubMed2006

A highly informative, multiplexed assay for the indirect detection of hemophilia A using five-linked microsatellites.

BACKGROUND: Hemophilia A is a severe bleeding disorder caused by almost 1000 different known mutations in the F8C gene. Direct mutation analysis is sometimes difficult for this disorder. When a mutation cannot be found, linkage analysis can be used for prenatal and carrier diagnosis. AIM: To develop a rapid and effective system for carrier detection and prenatal diagnosis of hemophilia A based on a single-multiplexed polymerase chain reaction (PCR) reaction utilizing five microsatellite markers. PATIENTS AND METHODS: Two intronic microsatellites and three other markers flanking the factor VIII gene were ascertained, and primers were designed for multiplex PCR amplification. A kindred with Hemophilia A was tested for linkage using the panel of primers, and informativity in the general population was ascertained by testing 50 unrelated females. RESULTS: Co-amplification of all microsatellites was optimized using DNA extracted by standard methods. Rapid detection and sizing of products were carried out using an automated DNA sequencer. The combined microsatellite panel was informative in each of the kindreds tested, and in 100% of the 50 unrelated females (95% CI 94.2-100%). CONCLUSIONS: This method enables the indirect detection of hemophilia A for patients in whom mutations cannot be found, facilitating carrier testing and prenatal analysis. It is rapid and straightforward compared with many other published protocols, and offers a high degree of informativity.

Base Sequence↗

PubMed2005

Internalization and desensitization of the oxytocin receptor is inhibited by Dynamin and clathrin mutants in human embryonic kidney 293 cells.

Oxytocin (OT) has long been used as an uterotonic during labor management in women, and yet responses to OT infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of OT is not optimal. As with other G protein-coupled receptors, it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur, however, are unclear. Here we explore OTR internalization and desensitization in human embryonic kidney cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca(2+) calmodulin-dependant kinase II but was significantly reduced after transfection with dominant-negative mutant cDNAs of G protein-coupled receptor kinase 2, beta-Arrestin2, Dynamin, and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate, for the first time, the importance of the classical clathrin-mediated pathway during agonist-induced OTR internalization and desensitization.

Arrestin↗

PubMed2005

Leukocyte origin and profile in follicular aspirates at oocyte retrieval.

BACKGROUND: Follicular aspirates represent a snapshot in time of conditions within the follicle at oocyte retrieval in women undergoing in vitro fertilization and embryo transfer. This clinical material has been much investigated and yet its cellular composition remains unclear. In this study we investigated the origin and profile of leukocytes found within follicular aspirates. METHODS: We performed morphological and immunohistochemical analyses of follicular aspirates and peripheral blood obtained concurrently at oocyte retrieval. RESULTS: There was no correlation between erythrocyte and leukocyte numbers in follicular aspirates. The profile of leukocyte subtypes within follicular aspirates was variable and differed significantly from the peripheral circulation in a significant proportion of the analysed samples. A subset of follicular aspirates displayed a marked increase in monocytes/macrophages and an apparent concomitant reduction in polymorphonuclear leukocytes compared with peripheral blood. CONCLUSIONS: Leukocytes within follicular aspirates cannot be accounted for solely as a result of blood vessel damage during oocyte retrieval. The variation in leukocyte subtypes observed in some follicular aspirates may reflect a coordinated infiltration of these cells, characteristic of progressive inflammatory responses in other systems. The possibility that leukocyte variation is indicative of follicular maturation deserves further investigation due to its potential relevance in optimizing oocyte selection.

Adult↗

PubMed2005

A postmarketing surveillance study of the safety and efficacy of ReFacto (St Louis-derived active substance) in patients with haemophilia A.

This clinical trial evaluated the safety and efficacy of ReFacto (St Louis-derived active substance) in patients with severe or moderately severe haemophilia A over a period of 6 months or 50 exposure days (EDs), whichever occurred first. Sixty patients, 58 previously treated and two previously untreated, were enrolled into this study. This was an open-label, multicentre, postmarketing surveillance study in which patients received prophylaxis or on-demand treatment as determined by their doctor. Surgical prophylaxis was evaluated in seven patients requiring elective surgery. Thirty-two patients aged <1 to 66 years (median 19.5) received prophylaxis and 28 patients, aged 1-71 years (median 33.5), received on-demand treatment. The majority of patients had severe haemophilia A (FVIII:C < 2%): 84.4% in the prophylaxis group and 85.7% in the on-demand group. Prophylaxis with ReFacto was associated with a median of 6.7 bleeds per year (range: 0-37). The investigator's assessment of final outcome for prophylactic treatment was excellent or effective for 93.1% of patients. ReFacto resolved 92.8% of bleeds with one or two infusions. The investigator's assessment was excellent or good for 98.2% of bleeds treated with ReFacto. Haemostasis was achieved for all seven surgical cases and ReFacto gave an excellent or good response for each. The nature and incidence of adverse events was as expected and no new safety concerns emerged. One previously treated patient (PTP) developed a high-titre inhibitor (maximum 75 BU) and one minimally treated patient (MTP) developed a low-titre inhibitor while on the study but eventually achieved high titres (maximum 30 BU) after immune tolerance therapy was initiated with a plasma-derived FVIII product. One previously untreated patient (PUP) developed a transient low-titre inhibitor (0.4 BU). Other serious adverse events (SAEs) were unrelated to study treatment. There were no allergic events. The results of this study are consistent with the previously published ReFacto pivotal studies.

Adolescent↗

PubMed2005

Recombinant factor VIIa as an adjunctive therapy for patients requiring large volume transfusion: a pharmacoeconomic evaluation.

BACKGROUND: Acute haemorrhage requiring large volume transfusion presents a costly and unpredictable risk to transfusion services. Recombinant factor VIIa (rFVIIa) (NovoSeven, Novo Nordisk, Bagsvaard, Denmark) may provide an important adjunctive haemostatic strategy for the management of patients requiring large volume blood transfusions. AIMS: To review blood transfusion over a 12-month period and assess the major costs associated with haemorrhage management. A pharmoeconomic evaluation of rFVIIa intervention for large volume transfusion was conducted to identify the most cost-effective strategy for using this haemostatic product. METHODS: Audit and analysis of all patients admitted to Christchurch Public Hospital requiring > 5 units of red blood cells (RBC) during a single transfusion episode. Patients were stratified into groups dependent on RBC units received and further stratified with regard to ward category. Cumulative costs were derived to compare standard treatment with an hypothesized rFVIIa intervention for each transfusion group. Sensitivity analyses were performed by varying parameters and comparing to original outcomes. RESULTS: Comparison of costs between the standard and hypothetical model indicated no statistically significant differences between groups (P < 0.05). Univariate and multivariate sensitivity analyses indicate that intervention with rFVIIa after transfusion of 14 RBC units may be cost-effective due to conservation of blood components and reduction in duration of intensive area stay. CONCLUSION: Intervention with rFVIIa for haemorrhage control is most cost-effective relatively early in the RBC transfusion period. Our hypothetical model indicates the optimal time point is when 14 RBC units have been transfused.

Acute Disease↗

PubMed2003

A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface.

To investigate Yersinia pathogenicity and the evolutionary divergence of the genus, the effect of pathogenic yersiniae on the model organism Caenorhabditis elegans was studied. Three strains of Yersinia pestis, including a strain lacking pMT1, caused blockage and death of C. elegans; one strain, lacking the haemin storage (hms) locus, caused no effect. Similarly, 15 strains of Yersinia enterocolitica caused no effect. Strains of Yersinia pseudotuberculosis showed different levels of pathogenicity. The majority of strains (76 %) caused no discernible effect; 5 % caused a weak infection, 9.5 % an intermediate infection, and 9.5 % a severe infection. There was no consistent relationship between serotype and severity of infection; nor was there any relationship between strains causing infection of C. elegans and those able to form a biofilm on an abiotic surface. Electron microscope and cytochemical examination of infected worms indicated that the infection phenotype is a result of biofilm formation on the head of the worm. Seven transposon mutants of Y. pseudotuberculosis strain YPIII pIB1 were completely or partially attenuated; mutated genes included genes encoding proteins involved in haemin storage and lipopolysaccharide biosynthesis. A screen of 15 defined C. elegans mutants identified four where mutation caused (complete) resistance to infection by Y. pseudotuberculosis YPIII pIB1. These mutants, srf-2, srf-3, srf-5 and the dauer pathway gene daf-1, also exhibit altered binding of lectins to the nematode surface. This suggests that biofilm formation on a biotic surface is an interactive process involving both bacterial and invertebrate control mechanisms.

Animals↗

PubMed2002

Hydrophobic interaction ligand selection and scale-up of an expanded bed separation of an intracellular enzyme from Saccharomyces cerevisiae.

A prototype Streamline-Phenyl matrix was evaluated in a hydrophobic interaction mode for the direct recovery of alcohol dehydrogenase (ADH) from yeast cell homogenate. At 5% breakthrough of ADH, a yield of 100% was obtained for a dynamic expanded bed capacity of 240 U(ADH)/ml matrix with a purification factor of 9.2. This compared with a dynamic capacity of 3013 U(ADH)/ml matrix for the packed bed equivalent and a purification factor of 18. In both systems the purification factor was found to increase simultaneously with a decrease in yield as the load of homogenate or breakthrough of ADH was increased. The expanded bed mode of operation conferred considerable robustness with respect to process fouling. No loss in yield was seen over five cycles of repeat loading with an unclarified homogenate. By contrast the packed bed media showed a decrease in yield from 86 to 56% over the same period. Successful scale up of the expanded bed protocol for a 20% breakthrough was demonstrated over a fourfold increase in column diameter. The application of hydrophobic interaction chromatography mediated expanded bed adsorption and its scale-up is discussed in the context of large-scale operations.

Adsorption↗

PubMed2002

The identification and classification of 41 novel mutations in the factor VIII gene (F8C).

Hemophilia A is a bleeding disorder caused by a quantitative or qualitative deficiency in the coagulation factor VIII. Causative mutations are heterogeneous in nature and are distributed throughout the FVIII gene. With the exception of mutations that result in prematurely truncated protein, it has proved difficult to correlate mutation type/amino acid substitution with severity of disease. We have identified 81 mutations in 96 unrelated patients, all of whom have typed negative for the common IVS-22 inversion mutation. Forty-one of these mutations are not recorded on F8C gene mutation databases. We have analyzed these 41 mutations with regard to location, whether or not each is a cross-species conserved region, and type of substitution and correlated this information with the clinical severity of the disease. Our findings support the view that the phenotypic result of a mutation in the FVIII gene correlates more with the position of the amino acid change within the 3D structure of the protein than with the actual nature of the alteration.

Animals↗

PubMed2002

Total cortisol levels are reduced in the periovulatory follicle of infertile women with minimal-mild endometriosis.

PROBLEM: To measure and compare concentrations of total and free glucocorticoids with oocyte fertilizing capacity in the follicular fluid (FF) of women with minimal-mild endometriosis and tubal damage. METHOD OF STUDY: Follicular fluid was collected from individual periovulatory follicles during oocyte retrieval for in vitro fertilization (IVF) in natural cycles. Total and free levels of cortisol and cortisone were measured using specific radioimmunoassays after chloroform extraction. RESULTS: Cortisol concentrations in women with minimal-mild endometriosis were significantly lower compared with controls (women with tubal infective damage) (258 versus 328 nmol/L, P < 0.02). There was no correlation between total or free concentrations of cortisol or cortisone and the fertilization capacity of the oocyte. CONCLUSIONS: Total cortisol levels are lower in the follicles of women with endometriosis. Our findings provide further evidence of follicular dysfunction contributing to the subfertility associated with minimal-mild endometriosis.

Case-Control Studies↗

PubMed2002

The Activated Seven Lupus Anticoagulant (ASLA) assay: a new test for lupus anticoagulants (LAs). Evidence that some LAs are detectable only in extrinsic pathway-based assays.

Accurate and timely detection of lupus anticoagulants (LAs) is of diagnostic and prognostic importance due to the association of persistent LAs with thrombotic disease. In the present study, a sensitive and specific assay for LAs has been developed using recombinant activated factor VII to initiate in vitro coagulation. The Activated Seven Lupus Anticoagulant (ASLA) assay uses dilute brain-derived phospholipid in the screening test and a platelet neutralization procedure (PNP) in the confirmatory test. Tests are reported as ratios of patient clotting time to normal control clotting time and percentage correction by PNP assessed for abnormal ratios. Evaluation with 70 known LA-positive plasmas demonstrated higher detection rates than with individual assays from a wide range of commonly employed LA tests. The ASLA assay identified 61 of 70 (87%) known LAs, compared with 65.7% with the most sensitive of the other assays. The various LA assays were used to test 110 plasma samples from patients with thrombotic disease previously negative for LA. These experiments demonstrated that 18 of 110 (16.4%) contained LAs detectable only in extrinsic pathway-based assays, 10 of these by ASLA testing alone. ASLA testing showed high diagnostic precision and has the potential to make a significant contribution to LA detection.

Blood Coagulation Disorders↗

PubMed2002

Venous thrombosis following the use of intermediate purity FVIII concentrate to treat patients with von Willebrand's disease.

We describe four patients with von Willebrand's disease (VWD) who experienced venous thrombosis after treatment with an intermediate purity factor VIII (FVIII) concentrate (Haemate P3) was used to cover invasive or surgical procedures. Most patients had additional risk factors for venous thromboembolism (VTE) and it is difficult to be certain of the contribution of the concentrate to the VTE. In view of the recognised association between high factor VIII activity (FVIII:C) levels and VTE there is a physiological basis for this complication and it is important to consider this when administering FVIII containing concentrates to VWD patients.

Adult↗

PubMed2001

Out-of-hours emergency dental services--development of one possible local solution.

This paper describes the development of a local solution to the problem of the provision of out-of-hours dental care in Newcastle and North Tyneside in the north east of England. Focus groups were used to review the current provision of, and problems with, dental out-of-hours emergency provision. A consensus conference involving both general dental and medical practitioners, was subsequently used to develop possible alternative methods for the provision of out-of-hours emergency dental services. A centralised service delivered from a secure location in conjunction with general medical practitioners was developed which was dependent on a nurse-led triage. The linkage with NHS Direct may be an opportunity, in some locations, to integrate dental services more fully with other out-of-hours primary care services. The method described allowed a solution to be generated by practitioners themselves, thus giving ownership and acceptance to the chosen option.

Consensus Statements as Topic↗

PubMed2001

Novel monoamine transporter ligands reduce cocaine-induced enhancement of brain stimulation reward.

Six novel monoamine reuptake inhibitors were screened for their intrinsic effects on brain stimulation reward (BSR), as well as for their potential to reduce cocaine-induced reward-enhancement in that paradigm. Two of the compounds, nocaine-3B and 5-ara-74A (disubstituted piperidines) significantly reduced locus of rise (LOR), threshold measure of reward, at some doses. One compound, 1-RV-96A (a hybrid of the GBR and WIN-like agents) significantly reduced reward (increased LOR), but only at the highest dose tested. No effect of dose was found for MC9-20 (a GBR-like acyclic analogue of the N-bisarylmethoxyethyl-N'-phenylpropyl piperazine), nocaine-250B or 4-ara-42C (disubstituted piperidines). When cocaine (10 mg/kg, ip) and selected, hedonically neutral doses of novel compounds were combined, the following findings were obtained: MC9-20 (2.5 mg/kg, ip) showed a significant increase in cocaine-induced reward enhancement (0.2 log units or 53%). In contrast, nocaine-250B and 1-RV-96A (both at 10 mg/kg, ip) demonstrated a significant reduction (0.13 log units or 41%) in cocaine-induced reward enhancement (P<.01 and P<.05, respectively), as measured by changes in LOR. There were no differences in the maximum behavioral output (MAX) at either dose of each of the six drugs, or when selected doses were combined with cocaine. These results indicate that nocaine-250B and 1-RV-96A constitute two potential anticocaine compounds worthy of further behavioral and biochemical evaluation.

Animals↗

PubMed2001

Effects of hypoxia on isolated vessels and perfused gills of rainbow trout.

Local hypoxia dilates systemic and constricts pulmonary blood vessels in mammals without neural or humoral involvement. The direct effects of hypoxia on isolated vessels from bony fish have not been examined. In the present study, isolated vessels (efferent branchial artery, EBA; coeliacomesenteric artery, CMA; ventral aorta, VA; and anterior cardinal vein, ACV) from rainbow or steelhead trout (Oncorhynchus mykiss) were subjected to either passive load (resting tension) or contracted with a ligand or 50 mM KCl and then subjected to 60 min of hypoxia by N(2) administration and an additional 30 min of normoxia. All vessels were usually refractory to hypoxia under conditions of resting tension. EBAs, CMAs and VAs pre-contracted with a receptor-mediated ligand were all significantly relaxed by hypoxia and only VAs recovered significantly upon subsequent restoration of normoxia. In contrast, tension in all arteries pre-contracted with 50 mM KCl was elevated further in response to hypoxia. Conversely, ligand-contracted ACVs responded to hypoxia with a further increase in tension, whereas KCl-contracted ACVs relaxed. During apparently random 2-3-week periods EBA and CMA from steelhead and EBA from rainbow trout were hyper-reactive to hypoxia. Steelhead vessels responded to hypoxia with a rapid contraction that increased in magnitude over 3 days. These contractions were independent of pre-stimulation and they were dose-dependent upon PO(2). In isolated gills, hypoxic perfusate produced an immediate but transient elevation of resistance (R(GILL)) in all four gill arches. R(GILL) increased by as much as 30% of initial values and this response was unaltered upon a second hypoxic exposure. These studies demonstrate that isolated vascular segments of rainbow trout are indeed responsive to hypoxia and that these differential responses are vessel and tone dependent and the overall response may be altered by as yet unknown seasonal or environmental factors. Hypoxia-induced arterial relaxation is blocked by elevated external [K(+)], implicating alteration of transmembrane K(+) conductance and/or membrane potential in this depressor response. K(+)-channel closure or voltage-gated Ca(2+) influx cannot account for arterial vasoconstriction due to hypoxia during KCl contractions. Vascular responses to hypoxia could have a profound impact on local flow in vivo and could mediate ventilation-perfusion matching in the branchial circulation of fish.

Animals↗

PubMed2001

A rapid and cost-effective method for analysis of three common genetic risk factors for thrombosis.

A simple, rapid and cost-effective method for the analysis of three of the most widely screened genetic risk factors for thrombosis has been established. The protocol developed uses blood spots stored on filter paper (Guthrie spots) as well as DNA extracted from anticoagulated blood. The use of Guthrie spots taken at birth enables the retrospective study of patients who develop thrombotic complications without necessitating resampling. Following isolation of DNA, conventional fluorescence-labelled polymerase chain reaction (PCR) is performed using a thermostable DNA polymerase. Denatured, single-stranded PCR products are analysed on a semi-automated capillary-based genetic analyser, the data being stored electronically. This sensitive protocol obviates the need for endonuclease digestion and the associated gel running and documentation, and leads to a reduction in the recurrent costs of laboratory consumables.

Blood Specimen Collection↗

PubMed2001

Cardiovascular effects of the R- and S-enantiomers of ibutilide in conscious beagle dogs.

To support its class III antiarrhythmic candidacy, we examined the acute cardiovascular effects of ibutilide and its R- and S-enantiomers in conscious beagle dogs. Eight dogs were given stepped i.v. doses (0.01, 0.1, and 1.0 mg/kg) of these agents while monitoring changes in mean arterial pressure (MAP), heart rate (HR), and lead II ECG QTc length (index of class III activity). None of the treatments affected MAP and only R-ibutilide slightly increased HR. The R-isomer was also slightly more potent in prolonging QTc at the lowest dose, but its mean peak QTc change (+44 msec) overlapped those achieved with racemic (+37 msec) and S-ibutilide (+41 msec). Plasma drug analyses showed that total drug levels (sum of enantiomers) were similar with each agent, averaging 1, 12, and 170 ng/ml at 30 min after the low, middle, and high doses, respectively. Nearly equal enantiomer proportions were seen after racemic ibutilide, and no chiral inversion was seen after enantiomer administration. All treatments were well tolerated without enhanced ventricular ectopy. These data demonstrate that in conscious dogs, racemic, R-, and S-ibutilide similarly prolong QTc independent of appreciable cardiovascular changes, differential pharmacokinetics, or dysrhythmias, thereby helping to establish racemic ibutilide as the optimal developmental candidate.

Animals↗

PubMed2001

Elective surgery on factor VIII inhibitor patients using continuous infusion of recombinant activated factor VII: plasma factor VII activity of 10 IU/ml is associated with an increased incidence of bleeding.

We examined recombinant activated factor VII (rVIIa) administered by continuous infusion to eight patients with inhibitors to factor VIII, undergoing elective surgery. rVIIa was infused at a fixed rate of 16.5 microg/kg/h for a median of 13.5 days (range 1-26). There was effective haemostasis at this infusion rate in only one of two minor procedures and two of six major operations. Three patients experienced excessive bleeding despite plasma factor VII activity around 10 IU/ml. Serious bleeding occurred in two other patients caused by procedural errors unrelated to rVIIa and required re-operation. The median rVIIa clearance on day 1 was 57 ml/h/kg (range 18-100) and on day 3 was 100 ml/h/kg (range 61-200). Clearance on the final infusion day was not significantly different from day 3. The infusion did not induce pathological activation of the coagulation mechanism. The only thrombotic adverse events were two episodes of superficial thrombophlebitis of the infused vein in one subject. In conclusion, the 16.5 microg/kg/h infusion rate reliably achieves plasma factor VII activity levels of 10 IU/ml, but this level does not provide reliable haemostasis.

Adult↗