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Biomedical subjects

S J Thorpe

Publications and source records attributed to S J Thorpe.

At least 19 recordsLinked to original sources
PubMed2003

Clonal analysis of a human antimouse antibody (HAMA) response.

Circulating human antimouse antibodies (HAMAs) directed to mouse immunoglobulin G (IgG) are clinically significant, compromising mouse antibody therapy and imaging, and interfering in immunological assays. To investigate the HAMA response, 20 stable cell lines secreting human monoclonal antibodies reactive with mouse IgG were established from a donor with a history of exposure to mice. Their subclass and domain specificities were established by solid-phase binding, indirect haemagglutination assays and immunoblotting, using Igs of known subclass and Ig fragments. The heavy-chain variable region gene usage was determined for 12 HAMAs. Eight HAMAs were IgM, 11 HAMAs were IgG4 and one HAMA was IgG1, indicating an IgG4-dominated response. All of the IgG HAMAs reacted with epitopes present on the Fc portion; one was subclass-specific, nine were subclass-restricted and two were pan-IgG-reactive. Measurement of their affinities gave dissociation constants typically in the nanomolar range. Seven and five HAMAs were derived from variable heavy-chain 3 (VH3) and VH1 gene segments, respectively. The IgG HAMAs used different VH segments to the IgM HAMAs. JH regions were coded by JH4 in eight HAMAs. DH segment usage appeared to be restricted in the IgM HAMAs. Two IgG HAMAs were clonally related. These monoclonal HAMAs are potentially useful as reagents for detecting mouse IgG and as reference reagents for the investigation of the HAMA response in patients undergoing mouse monoclonal antibody therapy and for the investigation of the influence of HAMAs on immunodiagnostic tests.

Amino Acid Sequence↗
PubMed2003

Competitive enzyme-linked immunoassay of monoclonal immunoglobulin G anti-D preparations.

The development of monoclonal immunoglobulin G (IgG) anti-D for prophylaxis necessitates estimation of their potency in terms of their red cell-binding ability, but it is unclear which quantification methodology is most suitable for this. The aim of this study was to assess 50 monoclonal anti-D from the 4th International Workshop for quantitative and qualitative binding to red cells in a competitive enzyme-linked immunoassay (EIA) in which varying amounts of monoclonal antibodies (MoAbs) and a constant amount of biotinylated monoclonal anti-D (BRAD-5) compete for red cell binding. The potencies of the MoAb were estimated against the International Reference Preparation (IRP) for anti-D Ig. Two MoAbs as supplied were insufficiently inhibitory of biotinylated BRAD-5 binding to be quantified; the potencies of the remainder ranged from 4 to 343 IU mL-1 and included 11 MoAbs showing dose-responses that were nonparallel to those of other MoAb and the IRP. Estimation of the concentration of antibody in the supernatants by radial immunodiffusion ranged from 0.5 to 61 micro g mL-1, giving specific activities of <1-24 IU micro g-1. The results show that competitive EIA is suitable for quantitating most monoclonal anti-D for development and quality control purposes, regardless of their D epitope reactivity.

Antibodies, Monoclonal↗
PubMed2003

Batches of intravenous immunoglobulin associated with adverse reactions in recipients contain atypically high anti-Rh D activity.

BACKGROUND AND OBJECTIVES: The presence of anti-Rh D in intravenous immunoglobulin (IVIG) products has been claimed to be associated with adverse reactions in recipients. There is currently no regulatory specification to control the level of anti-D in IVIG products and it is unclear what this should be. Two reports of haemolysis occurring in recipients of IVIG manufactured from US plasma provided a rare opportunity to investigate whether high anti-D levels could have induced the haemolysis. MATERIALS AND METHODS: We developed a direct microtitre plate haemagglutination method suitable for screening IVIG products and starting plasma pools for haemagglutinating activity. RESULTS: Of 101 batches of IVIG tested, six were found to contain specific anti-D. Four of these batches had anti-D titres ranging from 64 to 256 (including the two batches each associated with a report of haemolysis) and could be linked, in each case, to a starting plasma pool also positive for anti-D. CONCLUSIONS: Our results show that IVIG products can contain appreciable anti-D levels. To avoid potential problems in recipients, we propose an anti-D titre of 8 as the maximum permissible limit of anti-D in IVIG products for batch acceptance and release. The availability of a reference preparation is essential for control of this proposed requirement.

Blood Cells↗
PubMed2002

International collaborative study to evaluate methods for quantification of anti-D in immunoglobulin preparations.

BACKGROUND AND OBJECTIVES: The disadvantages of autoanalyser methodology for anti-D potency estimation have prompted the search for an alternative reference method. The aim of this study was to carry out a direct comparison of autoanalyser methodology, competitive enzyme-linked immunoassay (EIA) and flow cytometry. MATERIALS AND METHODS: The anti-D potencies of nine immunoglobulin preparations were estimated against the World Health Organization (WHO) International Reference Preparation for anti-D immunoglobulin, using the three different assay methods described above, in an international collaborative study involving 18 laboratories. RESULTS: No significant differences in potency estimates for five of nine samples were identified using the three different methods. For six of nine samples the mean potency estimates obtained using competitive EIA lay between those of the autoanalyser and flow cytometry. Flow cytometry gave the lowest estimates for seven of nine samples. The range of intralaboratory variability, expressed as percentage geometric coefficients of variation (% gcv) and excluding extreme values, was as follows: autoanalyser (eight laboratories), 0.4-17.0; competitive EIA (12 laboratories), 2.6-34.8; and flow cytometry (eight laboratories), 2.9-30.0. The range of interlaboratory variability (expressed as % gcv) was: autoanalyser (eight laboratories, eight samples), 10.8-17.6; competitive EIA (12 laboratories, nine samples), 10.3-17.3; flow cytometry (nine laboratories, eight samples), 6.2-16.1. CONCLUSIONS: The study did not show clear superiority of one method over the others. Both competitive EIA and flow cytometry are acceptable alternatives to autoanalyser methodology for polyclonal anti-D potency estimation. For monoclonal anti-D, further work is necessary to determine the most appropriate method for potency testing.

Agglutination Tests↗
PubMed2001

The time course of visual processing: from early perception to decision-making.

Experiments investigating the mechanisms involved in visual processing often fail to separate low-level encoding mechanisms from higher-level behaviorally relevant ones. Using an alternating dual-task event-related potential (ERP) experimental paradigm (animals or vehicles categorization) where targets of one task are intermixed among distractors of the other, we show that visual categorization of a natural scene involves different mechanisms with different time courses: a perceptual, task-independent mechanism, followed by a task-related, category-independent process. Although average ERP responses reflect the visual category of the stimulus shortly after visual processing has begun (e.g. 75-80 msec), this difference is not correlated with the subject's behavior until 150 msec poststimulus.

Adult↗
PubMed2001

Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR.

The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.

Binding Sites↗
PubMed2001

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity.

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.

Catalysis↗
PubMed2001

Enhanced efficacy of anti-D immunoglobulin for treating ITP is not explained by higher immunoglobulin polymer content.

Several reports have suggested that low dose anti-D immunoglobulin is superior to high dose immunoglobulin for treatment of idiopathic thrombocytopenia purpura (ITP). However, some findings suggest that it is not the anti-D activity per se that is responsible for efficacy for treatment of ITP with anti-D immunoglobulin. Amongst alternative explanations for the mechanism of action is a relatively higher immunoglobulin polymer content of anti-D compared to other immunoglobulin products, which is more efficient in causing reticuloendothelial Fc receptor blockade. In order to investigate this we have evaluated the polymeric IgG content of anti-D and other immunoglobulin products. Different products showed considerable variation in immunoglobulin polymer content. There was no clear correlation between aggregate or dimer content and product type and anti-D as a class of product did not contain higher amounts of either dimer or aggregate compared to other products. Some manufacturers' products increased in polymer content on storage, but others did not show this effect. Therefore, higher immunoglobulin dimer and/or aggregate content cannot explain the increased efficacy of anti-D immunoglobulin for treatment of ITP. The role of polymeric Ig in efficacy for treatment of ITP is unclear.

Biopolymers↗
PubMed2001

Detection of animals in natural images using far peripheral vision.

It is generally believed that the acuity of the peripheral visual field is too poor to allow accurate object recognition and, that to be identified, most objects need to be brought into foveal vision by using saccadic eye movements. However, most measures of form vision in the periphery have been done at eccentricities below 10 degrees and have used relatively artificial stimuli such as letters, digits and compound Gabor patterns. Little is known about how such data would apply in the case of more naturalistic stimuli. Here humans were required to categorize briefly flashed (28 ms) unmasked photographs of natural scenes (39 degrees high, and 26 degrees across) on the basis of whether or not they contained an animal. The photographs appeared randomly in nine locations across virtually the entire extent of the horizontal visual field. Accuracy was 93.3% for central vision and decreased almost linearly with increasing eccentricity (89.8% at 13 degrees, 76.1% at 44.5 degrees and 71.2% at 57.5 degrees ). Even at the most extreme eccentricity, where the images were centred at 70.5 degrees, subjects scored 60.5% correct. No evidence was found for hemispheric specialization. This level of performance was achieved despite the fact that the position of the image was unpredictable, ruling out the use of precued attention to target locations. The results demonstrate that even high-level visual tasks involving object vision can be performed using the relatively coarse information provided by the peripheral retina.

Adult↗
PubMed2001

Is it a bird? Is it a plane? Ultra-rapid visual categorisation of natural and artifactual objects.

Visual processing is known to be very fast in ultra-rapid categorisation tasks where the subject has to decide whether a briefly flashed image belongs to a target category or not. Human subjects can respond in under 400 ms, and event-related-potential studies have shown that the underlying processing can be done in less than 150 ms. Monkeys trained to perform the same task have proved even faster. However, most of these experiments have only been done with biologically relevant target categories such as animals or food. Here we performed the same study on human subjects, alternating between a task in which the target category was 'animal', and a task in which the target category was 'means of transport'. These natural images of clearly artificial objects contained targets as varied as cars, trucks, trains, boats, aircraft, and hot-air balloons. However, the subjects performed almost identically in both tasks, with reaction times not significantly longer in the 'means of transport' task. These reaction times were much shorter than in any previous study on natural-image processing. We conclude that, at least for these two superordinate categories, the speed of ultra-rapid visual categorisation of natural scenes does not depend on the target category, and that this processing could rely primarily on feed-forward, automatic mechanisms.

Adult↗
PubMed2001

Rate coding versus temporal order coding: what the retinal ganglion cells tell the visual cortex.

It is often supposed that the messages sent to the visual cortex by the retinal ganglion cells are encoded by the mean firing rates observed on spike trains generated with a Poisson process. Using an information transmission approach, we evaluate the performances of two such codes, one based on the spike count and the other on the mean interspike interval, and compare the results with a rank order code, where the first ganglion cells to emit a spike are given a maximal weight. Our results show that the rate codes are far from optimal for fast information transmission and that the temporal structure of the spike train can be efficiently used to maximize the information transfer rate under conditions where each cell needs to fire only one spike.

Animals↗
PubMed2000

A competitive enzyme-linked immunoassay using erythrocytes fixed to microtitre plates for anti-D quantitation in immunoglobulin products.

BACKGROUND AND OBJECTIVES: The batch control of anti-D immunoglobulin for prevention of haemolytic disease of the newborn necessitates assessment of its potency. Anti-D quantitation is usually performed using automated haemagglutination methodology although this can only be carried out in specialist centres. The aim of this study was to develop a simple and robust assay for anti-D quantitation. METHODS: We developed a competitive enzyme-linked immunoassay (EIA) in which unlabelled anti-D immunoglobulin and a biotinylated monoclonal anti-D compete for red cell binding. Binding of biotinylated anti-D is detected using an alkaline-phosphatase-labelled avidin preparation. The assay is conveniently carried out using erythrocytes fixed to microtitre plates. RESULTS: The competitive EIA was specific for anti-D activity, highly reproducible and showed good correlation with manufacturers' potency estimates using automated haemagglutination. The assay was quick and simple to perform using freshly prepared or stored plates, and the biotinylated monoclonal anti-D could be lyophilized in ampoules for distribution as a standardized reagent. CONCLUSIONS: The competitive EIA described can be used for the specific quantitation of anti-D and provides a robust alternative method to automated haemagglutination.

Antibodies, Monoclonal↗
PubMed2000

National pathfinder survey of dental caries prevalence and treatment needs in The Gambia.

OBJECTIVE: The aim of this study was to assess dental caries status and treatment needs in The Gambia for the purpose of national planning. MATERIALS AND METHODS: 1,235 subjects were obtained with a multi-stage stratified random sampling technique. Clinical examinations were carried out using the criteria suggested in WHO Basic Methods (WHO 1987). The WHO J2 software programme was used for data analysis. RESULTS: More than half of the subjects had caries. Mean DMFT increased from 1.7 in the 7-year-old group to 8.8 in 65-99-year-olds. Mean DT peaked at 5.1 in the 30-34-year-old group before declining to 3.2 among the 65-99-year-olds. Mean filled teeth (FT) was zero in all ages. Missing teeth (MT) increased from 0.1 in 13-year-olds to 5.6 among the 65-99-year group. Mean DMFT was 2.3, 2.8, and 6.6 in 12-year-olds, 15-year-olds and 35-44-year-olds respectively. Unmet treatment need (DT/DMFT) was 90-100% in subjects below 25 years. Need for dental extraction increased from 15% to 63% in 65-99-year-olds. Between 0.3 and 2.8 teeth required extraction, and 46-79% of subjects needed conservative treatment. Dental auxiliaries should be trained to carry out extractions and fillings using the atraumatic restorative treatment (ART).

Adolescent↗
PubMed2000

National pathfinder survey of periodontal status and treatment needs in The Gambia.

OBJECTIVE: The aim of the pathfinder survey was to assess the periodontal status and treatment needs in The Gambia. METHODS: A multistage stratified random sampling technique was used to obtain a sample of 1,235 (or 0.1% of the national population). The study was conducted according to the World Health Organisation's criteria (WHO, 1987). The WHO J2 software programme was used to analyse the data. RESULTS: Not more than 12% of subjects in any age group had healthy periodontal tissues. Two to three sextants were healthy among those under the age of 35 years. Shallow (4-5 mm) pockets were present in all ages. However, less than one sextant was involved among subjects younger than 25 years. Despite the high prevalence of pockets, few sextants were edentulous. The majority of subjects needed oral hygiene instructions and oral prophylaxis. Of the subjects between the ages of 8 and 29 years, 5-28% needed complex treatment in only half of a sextant. Similarly, 38% and 80% of older subjects needed complex treatment in 0.8-1.9 sextants. CONCLUSION: A national oral health plan in The Gambia should focus on health education and provision of oral prophylaxis by trained auxiliary health care workers. In conclusion, there is a need to develop a functional district oral health services system in The Gambia.

Adolescent↗
PubMed1999

A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins.

Bacteriophage T5 5'-->3' exonuclease is a member of a family of sequence related 5'-nucleases which play an essential role in DNA replication. The 5'-nucleases have both exonucleolytic and structure-specific endo-nucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of a structure-specific single cleavage assay for this enzyme which uses a 5'-overhanging hairpin substrate. The products of DNA hydrolysis are characterised by mass spectrometry. The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'-->3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 10(15). The catalytic assay has been extended to three mutants of T5 5'-->3' exonuclease, K83A, K196A and K215A. Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency. All three lysines contribute to ground state binding of the substrate. In addition, K83 plays a significant role in the chemical reaction catalysed by this enzyme. Possible roles for mutated lysine residues are discussed.

Amino Acid Substitution↗
PubMed1999

Patient-controlled sedation using propofol: randomized, double-blind dose refinement.

This double-blind, randomized trial compared the onset of sedation with two patient-controlled sedation regimens, allowing a maximum of 16 or 25 mg min-1 propofol. Forty fit young patients presenting for elective surgery were asked to try to put themselves to sleep using the system. Onset times of sedative effect, slurred speech and amnesia were recorded. All patients achieved satisfactory sedation, and none became oversedated. Patients receiving 16 mg min-1 propofol were not reliably sedated within 5 min and took significantly longer to develop slurred speech and amnesia (P < 0.01 for both). We conclude that this maximum infusion rate does not produce amnesia or sedation rapidly enough to be clinically useful. A maximum infusion rate of 25 mg min-1 allowed rapid sedation in all patients without oversedation and may be an acceptable compromise between efficacy and safety.

Adolescent↗
PubMed1999

Evaluation of a panel of human monoclonal antibodies to D and exploration of the synergistic effects of blending IgG1 and IgG3 antibodies on their in vitro biologic function.

BACKGROUND: The D immunoprophylaxis program has successfully reduced the incidence of Rh hemolytic disease of the newborn (HDN), but it has also reduced the availability of plasma-derived polyclonal anti-D, which constitutes the current therapeutic product. Human monoclonal anti-D from hybridoma cell lines may be an acceptable alternative, and clinical efficacy of each anti-D is being evaluated in several centers. STUDY DESIGN AND METHODS: This study represents the largest assessment (outside of the International Workshops) of human D monoclonal antibodies for potential therapeutic use. The in vitro biologic activity and immunologic and serologic reactivity of a coded panel of 20 D antibodies (THERAD) was investigated. The bioassays used were lymphocyte (K-cell) antibody-dependent cell-mediated cytotoxicity (ADCC), monocyte ADCC, and monocyte chemiluminescence, which together reflect the processes involved in antibody-coated red cell destruction in vivo. From this panel, six antibodies (THERADs 14, 19, 22, 23, 27, and 28, comprising 3 IgG1 and 3 IgG3 D monoclonal antibodies) were further selected to investigate the effects of blending in the three bioassays. RESULTS: Several THERAD blends displayed greater activity than their component parts, in the range of 6 to 124 percent. There was no evidence to suggest functional blocking effects with this restricted panel of antibodies. CONCLUSION: The THERAD blends containing both IgG1 and IgG3 anti-D appeared to be the most functionally active, as did blends containing antibodies to two distinct D epitopes. This in vitro evidence has important implications for the future formulation of an effective monoclonal preparation for the prevention of Rh HDN.

Antibodies, Anti-Idiotypic↗