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S Townsend

Publications and source records attributed to S Townsend.

At least 19 recordsLinked to original sources
PubMed2006

Optimizing recombinant antibody function in SPR immunosensing. The influence of antibody structural format and chip surface chemistry on assay sensitivity.

BACKGROUND: Recombinant antibody fragments are valuable tools for SPR-based detection of small molecules such as illicit drugs. However, the multiple structural formats of recombinant antibody fragments are largely uncharacterised with respect to their respective performance in SPR sensing. We have expressed a model anti-M3G antibody in both scFv and chimeric Fab formats to examine its sensitivity and binding profiles in a microplate immunoassay format and Biacore. We have further examined the influence of scFv multimerisation, Fab constant region stability and SPR chip surface coating chemistry, on anti-hapten SPR assay development. RESULTS: Under optimised competition ELISA conditions, the anti-M3G scFv was found to have an IC(50) value of 30 ng/ml, while the most stable Fab construct exhibited an IC(50) value of 2.4 ng/ml. In SPR competition assay on an M3G-OVA-coated SPR chip surface, the two constructs again differed in sensitivity, with IC(50) values of 117 and 19 ng/ml for the scFv and Fab, respectively (the scFv also exhibiting poor linearity of response). However, when the SPR chip surface was directly coated with M3G, both antibody constructs exhibited good linearity of response, similar high sensitivity IC(50) values (scFv 30 ng/ml, Fab 14 ng/ml) and high reproducibility (50 effective regenerations for M3G-OVA, 200 for M3G direct). During SPR assay development it was noticed that scFv and Fab constructs gave differing off-rate profiles. Subsequent HPLC, ELISA and electrophoretic analyses then confirmed that a portion of the scFv population multimerises. Bivalent scFv was found to profoundly affect the dissociation curve for scFv in stringent SPR kinetic analyses, leading to a 40-fold difference in calculated off-rate values (Fab off rate 4.7 x 10(-3)S(-1), scFv off rate 1.03 x 10(-2)S(-1)). CONCLUSION: The structural format of recombinant antibody fragments and chip functionalisation methodology can both profoundly affect the function of anti-M3G SPR assay, with direct coating and Fab format proving to be optimal. The confirmation of scFv multimerisation and resulting changes in SPR kinetics profile, in comparison with a Fab, further suggest that caution must be taken in the interpretation of SPR sensorgrams, which are commonly used in the 'affinity ranking' of scFv panels in which the extent of dimerisation in each sample is unknown.

Animals↗
PubMed2005

A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction.

In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

Animals↗
PubMed2001

XRCC2 is a nuclear RAD51-like protein required for damage-dependent RAD51 focus formation without the need for ATP binding.

The human XRCC2 gene was recently identified by its ability to complement a hamster cell line, irs1, which is sensitive to DNA-damaging agents and shows genetic instability. The XRCC2 protein is highly conserved in mammalian species and has structural features, including a putative ATP-binding domain (P-loop), consistent with membership of the RecA/RAD51 family of recombination-repair proteins. We show that a hybrid XRCC2-green fluorescent protein, which was found to be functional by complementation, localizes to the nucleus. We have established a functional link between XRCC2 and RAD51 by looking at damage-dependent RAD51 focus formation in the irs1 cell line. Little or no formation of RAD51 foci occurred in irs1. This effect was specific to the loss of XRCC2 because transfection of the gene into irs1 restored normal levels of focus formation. Surprisingly, XRCC2 genes carrying site-specific mutations in P-loop residues were found to be able to complement the XRCC2-deficient irs1 line for a number of different end points. We conclude that XRCC2 is important in the early stages of homologous recombination in mammalian cells to facilitate RAD51-dependent recombination repair but that it does not make use of ATP binding to promote this function.

Adenosine Triphosphate↗
PubMed2000

Oral dietary supplements in pre- and postoperative surgical patients: a prospective and randomized clinical trial.

It has been suggested that the routine provision of oral dietary supplements (ODS) in postoperative surgical patients is of benefit in terms of morbidity and length of hospital stay. The aim of this study was to evaluate the effects of both pre- and postoperative ODS in patients undergoing an elective laparotomy. Patients requiring elective major gastrointestinal surgery were prospectively randomized into one of four groups: Group I received ODS in addition to normal diet both pre- and postoperatively, Group II were given ODS in the preoperative period only, Group III received ODS only in the postoperative period, and Group IV did not receive any supplements. Assessments of nutritional status, voluntary food intake, weight loss, serum albumin, morbidity and mortality, anxiety and depression, and postoperative activity levels were performed, and comparisons made between the groups. One hundred patients were included in the study. The mean daily energy intake from preoperative ODS was 507 +/- 140 kcal, significantly more than the 252 +/- 195 kcal in the postoperative period (P < 0.001). The postoperative voluntary food intake in patients receiving ODS was not significantly different from that in patients receiving normal diet alone (1090 versus 1268 kcal, 46.2 versus 49.1 g protein, P > 0. 05). All groups demonstrated an overall weight loss, with no significant differences between the groups, and there was no demonstrable effect on clinical outcome. At 6 mo postoperatively there were no differences between the study groups in terms of levels of activity. These results suggest that the routine use of perioperative ODS in well-nourished patients undergoing major gastrointestinal surgery confers no clinical or functional benefit.

Adult↗
PubMed1999

The effect of acupuncture on patients with rheumatoid arthritis: a randomized, placebo-controlled cross-over study.

OBJECTIVE: Acupuncture is commonly used by patients with chronic painful musculoskeletal disorders. There are, however, few well-designed studies of its efficacy. This paper describes a randomized placebo-controlled cross-over design to evaluate acupuncture as a useful treatment adjunct in the management of patients with rheumatoid arthritis (RA). METHODS: Sixty-four patients were centrally randomized from a hospital-based rheumatology out-patient clinic. Fifty-six patients were suitable for study, all were on second-line therapy and aged 18-75 yr. There had been no change in therapy for the preceding 3 months. Patients who had previous acupuncture, anticoagulation, fear of needles or infection were excluded. Single-point (Liver 3) acupuncture or placebo was given with an intervening 6 week wash-out period. The acupuncturist, patient and statistician were blinded as far as possible. The outcome measures included the inflammatory markers (erythrocyte sedimentation rate and C-reactive protein), visual analogue scale of pain, global patient assessment, 28 swollen and tender joint count, and a general health questionnaire. RESULTS: The results demonstrated no significant effect of treatment or period and no significant interaction between treatment and period for any outcome variable. No adverse effects were reported. CONCLUSION: Acupuncture of this type cannot be considered as a useful adjunct to therapy in patients with RA. Possible reasons why this is the case are discussed.

Acupuncture Therapy↗
PubMed1999

Does splanchnic ischemia occur in isolated neurotrauma? A prospective observational study.

OBJECTIVE: To characterize the incidence and severity of splanchnic ischemia, as defined by gastric tonometry, in patients with isolated severe head injury and to examine the relationship between cerebral hemodynamics and splanchnic ischemia. DESIGN: Prospective observational study. SETTING: Neurosurgical intensive care unit in a tertiary referral center. PATIENTS: Ten patients with severe neurotrauma. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The mean arterial pressure, intracranial pressure, and gastric mucosal P(CO2) measurements were recorded at 15-min intervals. Intramucosal pH was calculated every 3 hrs. All patients received stress ulcer prophylaxis. Nine patients received noradrenaline infusions to maintain a target cerebral perfusion pressure of 70 mm Hg. The mean baseline gastric mucosal P(CO2) and intramucosal pH were 38+/-10 torr and 7.38+/-0.1 pH units, respectively. Nine patients manifested low intramucosal pH during the study period. Gastric mucosal P(CO2) values ranged from 36 to 132 torr. Intramucosal pH measurements ranged from 6.9 to 7.47. The mucosal gap ranged from -12 to +93 torr (mean +/- SD, 17+/-17 torr). The pH gap ranged from -0.1 to +0.54 pH units (mean +/- SD, 0.14+/-0.11 pH units). There was no statistically significant relationship between cerebral hemodynamics, the use of inotropes, and gastric mucosal P(CO2), or intramucosal pH. CONCLUSIONS: Splanchnic ischemia (intramucosal pH, <7.3) occurs commonly in isolated neurotrauma, with a statistically nonsignificant trend toward development of mucosal ischemia with decreased cerebral perfusion.

Acidosis↗
PubMed1998

Infant nutrition program effectively prevents iron-deficiency anemia in a First Nations community.

Iron-deficiency anemia (IDA) has received relatively little attention in Canada, with no national public health initiatives even among high-risk infants. IDA has a high prevalence in First Nations children and has been shown to cause developmental delay. This study is a before/after prevalence survey studying the effect of a public health intervention, conducted on a First Nations reserve off the central coast of British Columbia (BC). We screened for IDA one cohort of infants born January 1993 to August 1994 and between the ages of 6 and 24 months. Twenty-five of a possible 37 infants were screened. We found 13 (52%) of the 25 had anemia, with a hemoglobin less than 100 g/L. The average hemoglobin was 98.9 +/- 19.2 g/L. The subsequent implementation of an infant nutrition program focused on educating parents and encouraging the use of iron-fortified formula for nonbreast-fed infants. In the year following program implementation, the cohort of infants born between September 1994 and September 1995 were screened for IDA when they were between the ages of 6 and 15 months. Twenty-four of 27 infants participated. Only one infant was anemic with a hemoglobin less than 100 g/L. The average hemoglobin was 116.6 +/- 11.6 g/L. The increase in the average hemoglobin and the decrease in the prevalence of anemia were both highly significant (p < .01). We judged our intervention to be very effective and would recommend similar programs for other First Nations communities.

Anemia, Iron-Deficiency↗
PubMed1995

The durability and distribution of glass fibres in the rat following intra-peritoneal injection.

Intra-peritoneal (IP) injection is being recommended as a means of assessing potential carcinogenicity of MMF following inhalation. Little is known of the behaviour of fibres in the peritoneal cavity or its relevance to the lung. This study considered both the biopersistence and the distribution of dose following IP injection of fibres. Biopersistence of fibres in the peritoneal cavity has been compared with that observed previously in the lung. Marked differences were found, with long fibres (> 20 microns) being more durable in the peritoneal cavity than in the lung. Breakage could not account for this finding, whereas differences in dissolution could. The behaviour of fibres and powders and their distribution in the peritoneal cavity following injection of different masses is reported. Distribution of dose depended on injection mass, with masses of < 1.5 mg showing even uptake onto the surfaces of the peritoneal organs, and higher masses resulting in the development of nodules of injection material, free in the peritoneal cavity, or loosely bound to the peritoneum. With fine powder, some clearance was observed over the first 48 h after IP injection, but not with fibres. The findings on both durability and distribution of dose following IP injection have implications on the justification for the use of IP injections in assessment of potential carcinogenicity of fibres following inhalation.

Animals↗
PubMed1994

The behavior of glass fibers in the rat following intraperitoneal injection.

Potential carcinogenicity of fibers is believed to be determined by three factors: the dose, dimensions and durability of the fibers concerned. Currently there is considerable debate on the appropriateness of using results from intraperitoneal (i.p.) injection studies to predict the potential carcinogenicity of airborne fibers following inhalation. For ip results to have any significance to potential inhalation hazards, there should be some relation between the biopersistence, dose, and dose distribution of fibers in the serosal cavity and in the lung. Preliminary results on the durability of one experimental glass fiber in the peritoneal cavity suggest differences in dissolution when compared with durability in the lung. In the lung, the diameters of the long fibers (> 20 microns) were observed to decline at a rate consistent with their exposure to a neutral pH environment. The diameter of shorter fibers declined much more slowly, consistent with exposure to a more acidic environment such as is found in the phagolysosomes of alveolar macrophages. In the peritoneal cavity all fibers, regardless of length, dissolved at the same rate as short fibers in the lung. The effect of dose on the distribution of fibers in the peritoneal cavity was investigated using similar experimental glass fibers and compared with that of a powder made from ground fibers. For both materials at doses up to 1.5 mg, material was taken up by the peritoneal organs roughly in proportion to their surface area. This uptake was complete 1-2 days after injection. At higher doses, the majority of the material in excess of this 1.5 mg formed clumps of fibers (nodules) which were either free in the peritoneal cavity or loosely bound to peritoneal organs. These nodules displayed classic foreign body reactions with an associated granulomatous inflammatory response. The findings on both durability in the peritoneal cavity and the presence of two distinct populations of material following i.p. injection have implications for the justification of the use of i.p. injections to assess potential carcinogenicity of fibers following inhalation.

Animals↗