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Biomedical subjects

A Gill

Publications and source records attributed to A Gill.

At least 37 records · Page 2Linked to original sources
PubMed1999

A three-dimensional in-vitro model for the study of peritoneal tumour metastasis.

Peritoneal metastasis is a frequent complication of gastrointestinal malignancy. We have developed a three-dimensional model of the human peritoneum that simulates the metastatic process in vitro. Peritoneal fibroblasts were incorporated into collagen lattices, allowed to contract, then overlaid with mesothelial cells. Scanning and transmission electron microscopy showed the model to have similar physical properties to human peritoneum. Mesothelial expression of the beta1 integrin family, the basement membrane proteins fibronectin, laminin, collagen types III and IV, and the cell adhesion molecules ICAM-1, VCAM-1 and PECAM were assessed and showed similar results to in vivo tissue. Gastrointestinal tumour cells seeded onto the model exhibited mesothelial adhesion, cell spreading and vesicle formation, and invasion of the mesothelial monolayer on scanning electron microscopy. Two distinct patterns of tumour cell growth were observed using light microscopy: a superficial spreading layer, and discrete invasive deposits. Invasion was accompanied by disruption of the mesothelial monolayer, degradation and re-orientation of the matrix, and rudimentary tumour cell differentiation. We believe the use of this in vitro peritoneal model will facilitate the study of the molecular mechanisms involved in the metastatic process.

Humans↗
PubMed1999

Phosphoenolpyruvate carboxylase kinase is a novel protein kinase regulated at the level of expression.

Phosphorylation of phosphoenolpyruvate carboxylase plays a key role in the control of plant metabolism. Phosphoenolpyruvate carboxylase kinase is a Ca2+-independent enzyme that is activated by a process involving protein synthesis in response to a range of signals in different plant tissues. The component whose synthesis is required for activation has not previously been identified, nor has the kinase been characterised at a molecular level. We report the cloning of phosphoenolpyruvate carboxylase kinase from the Crassulacean Acid Metabolism plant Kalanchoë fedtschenkoi and the C3 plant Arabidopsis thaliana. Surprisingly, phosphoenolpyruvate carboxylase kinase is a member of the Ca2+/calmodulin-regulated group of protein kinases. However, it lacks the auto-inhibitory region and EF hands of plant Ca2+-dependent protein kinases, explaining its Ca2+-independence. Its sequence is novel in that it comprises only a protein kinase catalytic domain with no regulatory regions; it appears to be the smallest known protein kinase. In K. fedtschenkoi, the abundance of phosphoenolpyruvate carboxylase kinase transcripts increases during leaf development. The transcript level in mature leaves is very low during the photoperiod, reaches a peak in the middle of the dark period and correlates with kinase activity. It exhibits a circadian oscillation in constant conditions. Protein kinases are typically regulated by second messengers, phosphorylation or protein/protein interactions. Phosphoenolpyruvate carboxylase kinase is an exception to this general rule, being controlled only at the level of expression. In K. fedtschenkoi, its expression is controlled both developmentally and by a circadian oscillator.

Amino Acid Sequence↗
PubMed1998

Surface plasmon resonance: a study of the effect of biotinylation on the selection of antibodies for use in immunoassays.

Surface plasmon resonance is a valuable optical phenomenon for monitoring biomolecular interactions in real time. In this project anti-mouse-Fc was coupled to the carboxymethyl dextran coating on the surface of a CM5 sensor chip (BIAcore) using amine coupling. Monoclonal antibodies (MAbs) to Luteinizing Hormone (LH) were then captured on this surface in the correct orientation for binding. LH (500 IU/l) was injected over the surface and the subsequent binding and dissociation events were monitored. The resulting optical response curves allowed fast analysis of the binding interactions of eight selected MAbs. It was possible to develop a two-site immunometric assay for LH using a pair of these MAbs. The effect of biotinylating the MAbs, using various biotin:antibody coupling ratios, on their subsequent binding to both LH and avidin conjugated alkaline phosphatase was also investigated. This approach has allowed rapid evaluation of the effect of changes in both reagent and reaction conditions on immunoassay performance and appears to be a valuable adjunct to immunosensor and immunoassay development.

Alkaline Phosphatase↗
PubMed1998

Bioprocess monitoring: an optical biosensor for rapid bioproduct analysis.

The use of an optical biosensor for rapid bioproduct analysis is described. The biosensor, which is sensitive to changes in the concentration of bioproduct at its biologically active surface, has been shown to provide concentration data within 10 s of sample addition to the device. This has been achieved through the use of linear regression analysis to extract information from the early part of the biosensor interaction profiles. The system has been used to monitor both the production and purification of antibody fragments expressed during batch fermentation of recombinant Escherichia coli. Data obtained using the biosensor have been used to provide real-time profiles describing the location of antibody fragments during bioprocessing. Biosensor data have also been compared with those obtained from ELISA, the traditional method of retrospective analyses of samples collected during bioprocessing.

Animals↗
PubMed1998

An optical biosensor for real-time chromatography monitoring: breakthrough determination.

The use of an optical biosensor for immunorecognition of protein products during affinity chromatography is discussed to provide rapid data describing the loading and subsequent breakthrough, followed by elution and fraction collection. The optical biosensor works by following in real-time the interaction of soluble ligate with an appropriate ligand attached to the optically active surface. The initial rate of interaction between soluble ligate and immobilized ligand has been shown to correlate well with ligate concentration. This method of analysis has also been shown to agree well with ELISA, the traditionally employed technique for immunoassay of protein products lacking, for example, catalytic activity. Forward prediction, using models of the breakthrough fitted to the real-time data, has enabled the column saturation point to be determined before it has been reached, thus enabling appropriate action to ensure minimal loss of protein product while improving column utilization efficiency. The biosensor, operated within a flow injection analysis regime, has been demonstrated to provide concentration data within 10 s, with a total assay turnaround of 30 s.

Animals↗
PubMed1998

Stability of a single-chain Fv antibody fragment when exposed to a high shear environment combined with air-liquid interfaces.

The effect of shear on the antigen binding activity of a recombinant scFv antibody fragment was investigated in the presence of air-liquid interfaces using a stirred vessel that was incompletely filled. Changes in binding activity of the scFv to its antigen were monitored using an optical biosensor which had been sensitized with hen egg lysozyme (the antigen). The biosensor response was used as a measure of scFv binding activity. In buffer solution (mean velocity gradient approximately 20,000 s-1), loss of binding activity followed a first-order model with a mean rate constant of 0.83 h-1. In unstirred buffer solution, no such loss was observed. Similarly, in sheared fermentation broth there was no loss of binding activity and protective effects were attributed to the antifoam PPG.

Biosensing Techniques↗
PubMed1998

Rapid monitoring of virus-like particles using an optical biosensor: a feasibility study.

Virus-like particles (VLPs) are multimeric proteins expressed by Saccharomyces cerevisiae. The particles are approximately 80 nm in diameter and they are used as a framework for a range of biological products; for example as carriers of viral antigens. Rapid monitoring of purified VLPs was investigated using an optical biosensor. The aim was to develop an assay which may be employed for real-time bioprocess monitoring of VLPs. Problems of mass transfer of analyte were overcome through selection of a planar biosensor surface, in preference to the traditional polymer-coated surface. To prolong the surface activity for interaction analysis, a sandwich assay was developed which involved the use of a secondary capture species. It was shown that VLP concentration in pure solution could be determined within 10 min.

Biosensing Techniques↗
PubMed1998

[The Monte Carlo method and parallel estimation in the drawing up of radiosurgery treatment plans].

PURPOSE: We investigated the practical application of a calculation algorithm based on the Monte Carlo method to stereotactic radiosurgery treatment planning. In radiosurgery, high dose gradients and the lack of electronic disequilibrium make high resolution matrices and high computing power and speed necessary to obtain accurate dose distribution. To date, the main obstacle to the wider-spread use of the Monte Carlo method has been the huge computing time necessary to obtain a dose distribution on current hardware. MATERIAL AND METHODS: In this project, developed within the ESPRIT program, funded by the European Union, a Parsytec CC (Cognitive Computing) computer was used with 9 processors (Power PC 604, 133 Mhz, RAM 64 Mb) with IBM AIX/EPX OS and availability for Fortran parallel codes compilation, connected to a PC for data input, results rendering, and dose distribution calculation with a conventional algorithm for comparison with the Monte Carlo code (an EGS4 user code). The module named Rapt Region Extractor performs data compression with an octree method without decreasing resolution, for RAM and computing time requirements to remain acceptable. A model of the 6 MV photon beam from Clinac 2100C Varian linear accelerator was devised, based on incident photon energy spectrum and, for each collimator dimension, on bidimensional dose distribution orthogonal to beam direction measured at SSd = SAD = 100 cm. RESULTS: Parallelization was carried out on event numbers, allowing a simulation speed to number of processor ratio close to unity. A new random number generator was used, capable of correctly running on the parallel architecture. The simulation procedure includes: 1) CT acquisition in DICOM 3.0 format, Analyze or with scanner; 2) Target delineation, treatment arc definition. 3) Dose calculation, with both conventional and Monte Carlo methods. 4) Dose distribution rendering on every transverse, sagittal or coronal planes overlapped in color wash on anatomical representation. Comparison between conventional and Monte Carlo algorithms were carried out on an anthropomorphic phantom and 10 real patients, with 2.5 mm anatomical resolution and standard deviation never exceeding 2%. A simulation with 10,000,000 events and 1% maximum variance can be run in 43'. When PTV is an homogeneous areas the differences between the two methods are around 5%, while when PTV is localized in dishomogeneous areas discrepancies reach 20% in the bone. CONCLUSIONS: In conclusion, the feasibility of direct simulation with the Monte Carlo method in radiosurgery has been demonstrated within time and hardware costs compatible with clinical practice.

Algorithms↗
PubMed1997

A randomized controlled trial of sedation in the critically ill.

A randomized controlled trial comparing: a) a combination of oral chloral hydrate and promethazine to b) a continuous intravenous midazolam infusion, for maintenance sedation in critically ill children, was carried out. The level of sedation was assessed four hourly using a specifically devized sedation scale. Forty-four children entered the study of whom two were subsequently excluded. The number of satisfactory assessments (desired and actual levels of sedation equal) was significantly greater in the chloral hydrate and promethazine group (Chi-squared P < 0.01; confidence intervals of the difference 0.06 to 0.20). The number of assessments at level 5 on the sedation scale (patient restless/ distressed) was significantly greater in the midazolam group (Chi-squared P < 0.05). The total number of satisfactory assessments in the two groups were only 61 and 48% respectively, suggesting that sedation can be considerably improved. Chloral hydrate and promethazine are more effective than midazolam as maintenance sedation in critically ill children. It is possible to prospectively study the efficacy of sedative drugs in critically ill children.

Administration, Oral↗
PubMed1996

Pathophysiologic role of alpha 4 integrins in the lung.

Evidence for a central role for the integrins alpha 4 beta 1 and alpha 4 beta 7 in leukocyte pathophysiology is rapidly accumulating. Five distinct alpha 4 mAbs, each able to block alpha 4-dependent adhesion in vitro, show beneficial effects in vivo in six different species, and in a wide variety of organ systems, including colon, lung, skin, neural tissue, pancreas, peritoneum, and the vessel wall. In particular, a clear role for these integrins in lung pathophysiology is implied on the basis of in vivo studies in four different species. Although several issues remain to be resolved, including the relative importance of alpha 4 beta 1 and alpha 4 beta 7, and the relative roles of their counterligands, VCAM1, fibronectin, and MAdCAM, the data argue that alpha 4 integrins will likely be critical to both the normal physiology and pathology of the lung in man. To this end, we (Adams, Lin, Lobb, and Gill, unpublished data) and others have generated peptidomimetic small molecule antagonists of VLA4 based on the connecting segment 1 (CS1) peptide sequence of fibronectin that are potent blockers of integrin adhesive function in vitro and show efficacy in vivo. We have found that our inhibitors are excellent blockers of both murine contact hypersensitivity, and of the LPR and AHR in the sheep allergic airways model (Abraham, Lobb, Adams, and Gill, unpublished data), and are therefore possible candidates for clinical intervention in human asthma. The use of the VCAM-Ig fusion protein as a probe for high-affinity alpha 4 integrins has further enhanced our understanding of alpha 4 integrin function in the lung. While integrin upregulation in vitro has been observed many times, and high affinity (as opposed to avidity) of integrins seen in vitro in several systems, in vivo proof of integrin upregulation to a high-affinity state has been difficult to obtain in the absence of selective probes. Our data provide key information in this regard and strongly argue not only that integrin upregulation does indeed occur in vivo, but also that it is in fact obligatory for the leukocyte pathologies we have examined to date. Further studies are clearly warranted to further examine mechanisms of action, and to confirm and extend these studies, both with the alpha 4 integrins and with other integrin families. In summary, our studies of alpha 4 integrins continue to provide novel insights into the pathophysiology of integrin function and into future directions for drug discovery.

Animals↗
PubMed1996

Analysis of kinetic data of antibody-antigen interaction from an optical biosensor by exponential curve fitting.

An optical biosensor system employing a resonant mirror (RM), with a stirred cuvette has been used to follow the interaction of a recombinant antibody fragment with its antigen, hen egg lysozyme. The data generated by the biosensor were analysed in order to determine the kinetic constants for the interaction using a linear transform (derivative analysis). For comparison the data were also analysed using an exponential curve fitting routine. It was demonstrated that the exponential curve fitting method produced results which were in agreement with the existing linear transform method. It was also shown that early fitting of the association phase response, using the exponential curve fitting routine between 0 and 70 s after sample addition, yielded sufficient information to provide a prediction of Kon. The potential use of the optical biosensor for the rapid monitoring of protein production and purification is discussed.

Animals↗
PubMed1996

Neurological outcome of severe cystic periventricular leukomalacia.

OBJECTIVE: To determine the neurological outcome of a cohort of premature babies having ultrasound diagnoses of severe cystic periventricular leukomalacia. METHODOLOGY: All neonatal intensive care unit admissions born at less than 35 weeks gestation or weighing less than 1500 g underwent serial cranial ultrasounds. Those developing severe bilateral cystic periventricular leukomalacia (12 patients) were then followed clinically to a mean age of 27.3 months. RESULTS: Ten of the 12 patients fulfilling strict ultrasound criteria survived. All had a major neurological handicap, all having spastic quadriparesis and visual impairments, with most suffering global developmental delay and epilepsy. CONCLUSIONS: Severe bilateral cystic periventricular leukomalacia results in major permanent handicap. It is currently impossible to identify most patients with this condition while they are being ventilated. Non-ultrasound techniques are needed to diagnose the condition earlier, and to give guidance to management.

Developmental Disabilities↗
PubMed1996

Risk-stratified nosocomial infection surveillance in a neonatal intensive care unit: report on 24 months of surveillance.

OBJECTIVE: To document the nosocomial infection rate in a single neonatal intensive care unit (NICU) in terms of patient workload and device utilization. METHODOLOGY: Nosocomial infections have been identified and documented by the methodology described by the National Nosocomial Infection Surveillance System (NNIS), Centres for Disease Control, Atlanta. In addition, antibiotic usage has been surveyed in the NICU and standardized measures of patient exposure to antibiotics stratified by birthweight and gestational age have been described. RESULTS: Overall nosocomial infection rates compared favourably with the published NNIS figures at 6.2 infections per 100 admissions or 4.8 per 1000 patient days. Infection rates were significantly higher in lower birthweight groups. Device-related infection rates in each birthweight cohort were also very close to published figures and varied less with birthweight group. Antibiotic exposure averaged 12% of total admission days, less than previously published data. CONCLUSIONS: The NNIS system is applicable to Australian NICU and provides an effective tool for monitoring infection episodes.

Anti-Bacterial Agents↗
PubMed1995

Kinetics of protein-protein interactions at the surface of an optical biosensor.

Methods based on the use of optical biosensors have recently become available to provide a convenient means of determining the rate and equilibrium constants for bimolecular interactions between immobilized ligands and soluble ligate molecules. However, the association data that these methods provide are not always accurately described by the expected pseudo-first-order reaction mechanism, particularly when the ligand is immobilized on a dextran matrix. We show that a better description of the association data, especially at higher ligate concentrations, is achieved with a double exponential function, indicating that at least two rate-limiting processes are involved. Various models are considered in order to explain these observations: the presence of two (or more) distinct populations of immobilized ligand; a change, possibly conformational, in the immobilized ligand before or after ligate binding; or the hindrance of ligate binding to immobilized ligand. We suggest that steric hindrance caused by ligate binding to the dextran-coated sensor surface seems the most likely explanation for the observed biphasic association kinetics and that the faster initial phase should be used in oder to determine association constants that can be compared to those in solution.

Antigen-Antibody Reactions↗