Biomedical subjects

B Vojnovic

Publications and source records attributed to B Vojnovic.

At least 19 recordsLinked to original sources

PubMed2006

Technique for measurement of fluorescence lifetime by use of stroboscopic excitation and continuous-wave detection.

A study of the practicality a simple technique for obtaining time-domain information that uses continuous wave detection of fluorescence is presented. We show that this technique has potential for use in assays for which a change in the lifetime of an indicator occurs in reaction to an analyte, in fluorescence resonance energy transfer, for example, and could be particularly important when one is carrying out such measurements in the scaled-down environment of a lab on a chip (biochip). A rate-equation model is presented that allows an objective analysis to be made of the relative importance of the key measurement parameters: optical saturation of the fluorophore and period of the excitation pulse. An experimental demonstration of the technique that uses a cuvette-based analysis of a carbocyanine dye and for which the excitation source is a 650 nm wavelength, self-pulsing AlGaInP laser diode is compared with the model.

Algorithms↗

PubMed2005

Advanced microscopy solutions for monitoring the kinetics and dynamics of drug-DNA targeting in living cells.

Many anticancer drugs require interaction with DNA or chromatin components of tumor cells to achieve therapeutic activity. Quantification and exploration of drug targeting dynamics can be highly informative in the rational development of new therapies and in the drug discovery pipeline. The problems faced include the potential infrequency and transient nature of critical events, the influence of micropharmacokinetics on the drug-target equilibria, the dependence on preserving cell function to demonstrate dynamic processes in situ, the need to map events in functional cells and the confounding effects of cell-to-cell heterogeneity. We demonstrate technological solutions in which we have integrated two-photon laser scanning microscopy (TPLSM) to track drug delivery in subcellular compartments, with the mapping of sites of critical molecular interactions. We address key design concepts for the development of modular tools used to uncover the complexity of drug targeting in single cells. First, we describe the combination of two-photon excitation with fluorescence lifetime imaging microscopy (FLIM) to map the nuclear docking of the anticancer drug topotecan (TPT) at a subset of DNA sites in nuclear structures of live breast tumor cells. Secondly, we demonstrate how we incorporate the smart design of a two-photon 'dark' DNA binding probe, such as DRAQ5, as a well-defined quenching probe to uncover sites of drug interaction. Finally, we discuss the future perspectives on introducing these modular kinetic assays in the high-content screening arena and the interlinking of the consequences of drug-target interactions with cellular stress responses.

Antineoplastic Agents↗

PubMed2004

Imaging protein-protein interactions in cell motility using fluorescence resonance energy transfer (FRET).

Protein-protein interactions and signal transduction pathways have traditionally been analysed using biochemical techniques or standard microscopy. Although invaluable in the delineation of protein hierarchy, these methods do not provide information on the true spatial and temporal nature of complex formation within the intact cell. Recent advances in microscopy have allowed the development of new methods to analyse protein-protein interactions at very high resolution in both fixed and live cells. The present paper provides a brief overview of using fluorescence resonance energy transfer to analyse directly molecular interactions and conformational changes in various proteins involved in the regulation of cell adhesion and motility.

Cell Adhesion↗

PubMed2003

Semi-automated software for the three-dimensional delineation of complex vascular networks.

The understanding of tumour angiogenesis is of great importance in cancer research, as is the tumour response to vascular-targeted drugs. This paper presents software aimed at aiding these investigations and other situations where linear or dendritic structures are to be delineated from three-dimensional (3D) data sets. This software application was written to analyse the data from 3D data sets by allowing the manual and semi-automated tracking and delineation of the vascular tree, including the measurement of vessel diameter. A new algorithm, CHARM, based on a compact Hough transform and the formation of a radial map, has been used to locate vessel centres and measure diameters automatically. The robustness of this algorithm to image smoothing and noise has been investigated.

Blood Vessels↗

PubMed2003

Low-dose studies of bystander cell killing with targeted soft X rays.

The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 microm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0-2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy). In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation. Damaged cells were distributed uniformly over the area of the dish scanned (approximately 25 mm2). However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.

Animals↗

PubMed2002

Investigating the cellular effects of isolated radiation tracks using microbeam techniques.

Studies of the effects of radiation at the cellular level have generally been carried out by exposing cells randomly to the charged-particle tracks of a radiation beam. Recently, a number of laboratories have developed techniques for microbeam irradiation of individual cells. These approaches are designed to remove much of the randomness of conventional methods and allow the nature of the targets and pathways involved in a range of radiation effects to be studied with greater selectivity. Another advantage is that the responses of individual cells can be followed in a time-lapse fashion and, for example, processes such as "bystander" effects can be studied clearly. The microbeam approach is of particular importance in mechanistic studies related to the risks associated with exposure to low fluences of charged particles. This is because it is now possible to determine the actions of strictly single particle tracks and thereby mimic, under in vitro conditions, exposures at low radiation dose that are significant for protection levels, especially those involving medium- to high-LET radiations. Overall, microbeam methods provide a new dimension in exploring mechanisms of radiation effect at the cellular level. Microbeam methods and their application to the study of the cellular effects of single charged-particle traversals are described.

Alpha Particles↗

PubMed2002

Upgrading of the Gray Laboratory soft X ray microprobe and V79 survival measurements following irradiation of one or all cells with a CK X ray beam of different size.

The X ray microprobe developed at the Gray Laboratory was originally designed to produce carbon K X rays (278 eV) by electron bombardment and focus them to a few hundred nanometers spot by using a circular diffraction grating with increasing line density (zone plate). The very fine focus achieved (< 0.25 micron radius spot) and the highly localised energy deposition of CK X rays (photoelectron range < 7 nm), represent unique tools to investigate modern radiobiological phenomena. Recent improvements have been directed to increase the dose rate (up to approximately 6 Gy.s-1 entrance dose averaged over a typical V79 cell) and to evaluate the possibility of using higher energy photons (AlK of 1.48 keV). The efficiency of the microprobe system has been tested by assessing the clonogenic potential of V79 cells irradiated with CK X ray beams of different sizes (5 and 0.25 microns radius) and investigating the relevance of the spatial distribution of cells for the bystander effect.

Animals↗

PubMed2001

Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability.

The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using intravital microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using N(omega)-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to approximately 160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.

Angiogenesis Inhibitors↗

PubMed2001

Automated counting of mammalian cell colonies.

Investigating the effect of low-dose radiation exposure on cells using assays of colony-forming ability requires large cell samples to maintain statistical accuracy. Manually counting the resulting colonies is a laborious task in which consistent objectivity is hard to achieve. This is true especially with some mammalian cell lines which form poorly defined or 'fuzzy' colonies, typified by glioma or fibroblast cell lines. A computer-vision-based automated colony counter is presented in this paper. It utilizes novel imaging and image-processing methods involving a modified form of the Hough transform. The automated counter is able to identify less-discrete cell colonies typical of these cell lines. The results of automated colony counting are compared with those from four manual (human) colony counts for the cell lines HT29, A172, U118 and IN1265. The results from the automated counts fall well within the distribution of the manual counts for all four cell lines with respect to surviving fraction (SF) versus dose curves, SF values at 2 Gy (SF2) and total area under the SF curve (Dbar). From the variation in the counts, it is shown that the automated counts are generally more consistent than the manual counts.

Automation↗

PubMed2001

Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic electrode in the p22 tumor.

Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO(2) readings lay in a range over twice that of sensor pO(2(5min)) values (-3.2 to 80 mm Hg and -0.1 to 34.8 mm Hg, respectively). However, there was no significant difference between the means +/- 2 SE of the median pO(2) values recorded by each instrument (11.0 +/- 3.3 and 8.1 +/- 1.9 mm Hg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means +/- 2 SE of the median pO(2) values reported by the electrode and sensor (1.7 +/- 0.9 and 2.9 +/- 0.7 mm Hg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO(2) values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO(2) values of 0.6 to 28.1 mm Hg.

Animals↗

PubMed2001

Low-dose hypersensitivity in Chinese hamster V79 cells targeted with counted protons using a charged-particle microbeam.

The Gray Laboratory charged-particle microbeam has been used to assess the clonogenic ability of Chinese hamster V79 cells after irradiation of their nuclei with a precisely defined number of protons with energies of 1.0 and 3.2 MeV. The microbeam uses a 1-microm silica capillary collimator to deliver protons to subcellular targets with high accuracy. The detection system is based on a miniature photomultiplier tube positioned above the cell dish, which detects the photons generated by the passage of the charged particles through an 18-microm-thick scintillator placed below the cells. With this system, a detection efficiency of greater than 99% is achieved. The cells are plated on specially designed dishes (3-microm-thick Mylar base), and the nuclei are identified by fluorescence microscopy. After an incubation period of 3 days, the cells are revisited individually to assess the formation of colonies from the surviving cells. For each energy investigated, the survival curve obtained for the microbeam shows a significant deviation below 1 Gy from a response extrapolated using the LQ model for the survival data above 1 Gy. The data are well fitted by a model that supports the hypothesis that radioresistance is induced by low-dose hypersensitivity. These studies demonstrate the potential of the microbeam for performing studies of the effects of single charged particles on cells in vitro. The hypersensitive responses observed are comparable with those reported by others using different radiations and techniques.

Animals↗

PubMed2001

A focused ultrasoft x-ray microbeam for targeting cells individually with submicrometer accuracy.

The application of microbeams is providing new insights into the actions of radiation at the cell and tissue levels. So far, this has been achieved exclusively through the use of collimated charged particles. One alternative is to use ultrasoft X rays, focused by X-ray diffractive optics. We have developed a unique facility that uses 0.2-0.8-mm-diameter zone plates to focus ultrasoft X rays to a beam of less than 1 microm diameter. The zone plate images characteristic K-shell X rays of carbon or aluminum, generated by focusing a beam of 5-10 keV electrons onto the appropriate target. By reflecting the X rays off a grazing-incidence mirror, the contaminating bremsstrahlung radiation is reduced to 2%. The focused X rays are then aimed at selected subcellular targets using rapid automated cell-finding and alignment procedures; up to 3000 cells per hour can be irradiated individually using this arrangement.

Dose-Response Relationship, Radiation↗

PubMed2000

Sweat gland function as a measure of radiation change.

BACKGROUND: Radiotherapy may result in dryness of the skin even when no other change can be detected. We describe a system for recording the electrical conductance of skin as a measure of sweat gland function. PATIENTS AND METHODS: In 22 normal volunteers close agreement was obtained between measurements obtained from comparable sites on both sides of the chest. Measurements were subsequently made in 38 patients treated by radiotherapy to one side of the chest for tumours of the breast or lung using one of five different fractionation schedules. Simultaneous readings were obtained from both sides of the chest with the non irradiated side acting as a control. RESULTS: A dose response relationship was demonstrated: five patients who received the equivalent total dose of 15 Gy in 2-Gy fractions showed no change in conductance. Sixteen out of 23 who received an equivalent total dose of 42-46 Gy in 2-Gy fractions had a greater than 22% reduction in mean skin conductance compared with that of the control areas despite the skin appearing normal in the large majority. Marked changes in skin conductance were seen after higher total doses. In a prospective study 18 women receiving breast irradiation underwent weekly readings during treatment. A mean reduction of 40% in skin conductance was noted by the end of the second week of treatment prior to any clinical evidence of radiation change. Skin conductance returned to normal in 44% of patients by 6 months. In the remainder, those patients who showed the greatest reduction in skin conductance during treatment demonstrated the least recovery. CONCLUSIONS: Changes in sweat gland function can be detected and quantified in skin which may otherwise appear normal. Differences may so be demonstrated between areas treated using different fractionation schedules and the method may be applied to the detection during radiotherapy of unusually sensitive patient.

Adult↗

PubMed2000

Critical energies for ssb and dsb induction in plasmid DNA by vacuum-UV photons: an arrangement for irradiating dry or hydrated DNA with monochromatic photons.

PURPOSE: Theoretical modelling techniques are often used to simulate the action of ionizing radiations on cells at the nanometre level. Using monoenergetic vacuum-UV (VUV) radiation to irradiate DNA either dry or humidified, the action spectra for the induction of DNA damage by low energy photons and the role of water and can be studied. These data provide inputs for the theoretical models. METHODS: Various combinations of monochromator, grating and VUV window have been used to obtain monochromatic photons from the 2 GeV electron synchrotron at the CLRC, Daresbury Laboratory. A sample chamber containing plasmid DNA is installed at the end of the beamline. The chamber can be evacuated or water can be introduced (as water vapour or humidified helium). In this way, DNA can be irradiated either dry or humidified. RESULTS: An arrangement for irradiating dry or humidified DNA using monoenergetic photons from 7 eV to 150 eV has been developed. At the energies used, exposure rates vary from about 5 x 10(10) to 3 x 10(12) photons cm(-2) s(-1) over a 1 cm2 sample area. At all but the lowest energies this is sufficient to produce significant levels of DNA damage in just a few minutes. The measured dose variation over the sample area is typically 30%, but this is reduced significantly using sample scanning techniques.

DNA Damage↗

PubMed2000

Critical energies for SSB and DSB induction in plasmid DNA by low-energy photons: action spectra for strand-break induction in plasmid DNA irradiated in vacuum.

PURPOSE: To measure action spectra for the induction of single-strand breaks (SSB) and double-strand breaks (DSB) in plasmid DNA by low-energy photons and provide estimates for the energy dependence of strand-break formation important for track-structure simulations of DNA damage. MATERIALS AND METHODS: Plasmid pMSG-CAT was irradiated as a monolayer, under vacuum, with 7 150eV photons produced by a synchrotron source. Yields of SSB and DSB were determined by the separation of the three plasmid forms by gel electrophoresis. RESULTS: The yields of SSB per incident photon increased from 1.4x 10(-15) SSB per plasmid per photon/cm2 at 7eV to 7.5 x 10(-14) SSB per plasmid per photon/cm2 at 150 eV. Direct induction of DSB was also detected increasing from 3.4 x 10(-17) DSB per plasmid per photon/cm2 at 7eV to 4.1 x 10(-15) DSB per plasmid per photon/cm2 at 150eV. When the absorption cross-section of the DNA was considered, the quantum efficiency for break formation increased over the energy range studied. Over the entire energy range, the ratio of SSB to DSB remained constant. CONCLUSIONS: These studies provide evidence for the ability of photons as low as 7 eV to induce both SSB and DSB. The common action spectrum for both lesions suggests that they derive from the same initial photoproducts under conditions where the DNA is irradiated in vacuum and a predominantly direct effect is being observed. The spectral and dose-effect behaviour indicates that DSB are induced predominantly by single-event processes in the energy range covered.

DNA↗

PubMed2000

Optical sensor-based oxygen tension measurements correspond with hypoxia marker binding in three human tumor xenograft lines.

Hypoxia has a negative effect on the outcome of radiotherapy and surgery and is also related to an increased incidence of distant metastasis. In this study, tumor pO(2) measurements using a newly developed time-resolved luminescence-based optical sensor (OxyLitetrade mark) were compared with bioreductive hypoxia marker binding (pimonidazole). Single pO(2) measurements per tumor were compared to hypoxia marker binding in tissue sections using image analysis. Both assays were performed in the same tumors of three human tumor lines grown as xenografts. Both assays demonstrated statistically significant differences in the oxygenation status of the three tumor lines. There was also a good correlation between hypoxia marker binding and the pO(2) measurements with the OxyLitetrade mark device. A limitation of the OxyLitetrade mark system is that it is not yet suited for sampling multiple sites in one tumor. An important strength is that continuous measurements can be taken at the same position and dynamic information on the oxygenation status of tumors can be obtained. The high spatial resolution of the hypoxia marker binding method can complement the limitations of the OxyLitetrade mark system. In the future, a bioreductive hypoxic cell marker for global assessment of tumor hypoxia may be combined with analysis of temporal changes in pO(2) with the OxyLitetrade mark to study the effects of oxygenation-modifying treatment on an individual basis.

Animals↗

PubMed1999

Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues.

The potential for tumor vascular-targeting by using the tubulin destabilizing agent disodium combretastatin A-4 3-0-phosphate (CA-4-P) was assessed in a rat system. This approach aims to shut down the established tumor vasculature, leading to the development of extensive tumor cell necrosis. The early vascular effects of CA-4-P were assessed in the s.c. implanted P22 carcinosarcoma and in a range of normal tissues. Blood flow was measured by the uptake of radiolabeled iodoantipyrine, and quantitative autoradiography was used to measure spatial heterogeneity of blood flow in tumor sections. CA-4-P (100 mg/kg i.p.) caused a significant increase in mean arterial blood pressure at 1 and 6 h after treatment and a very large decrease in tumor blood flow, which-by 6 h-was reduced approximately 100-fold. The spleen was the most affected normal tissue with a 7-fold reduction in blood flow at 6 h. Calculations of vascular resistance revealed some vascular changes in the heart and kidney for which there were no significant changes in blood flow. Quantitative autoradiography showed that CA-4-P increased the spatial heterogeneity in tumor blood flow. The drug affected peripheral tumor regions less than central regions. Administration of CA-4-P (30 mg/kg) in the presence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester, potentiated the effect of CA-4-P in tumor tissue. The combination increased tumor vascular resistance 300-fold compared with less than 7-fold for any of the normal tissues. This shows that tissue production of nitric oxide protects against the damaging vascular effects of CA-4-P. Significant changes in tumor vascular resistance could also be obtained in isolated tumor perfusions using a cell-free perfusate, although the changes were much less than those observed in vivo. This shows that the action of CA-4-P includes mechanisms other than those involving red cell viscosity, intravascular coagulation, and neutrophil adhesion. The uptake of CA-4-P and combretastatin A-4 (CA-4) was more efficient in tumor than in skeletal muscle tissue and dephosphorylation of CA-4-P to CA-4 was faster in the former. These results are promising for the use of CA-4-P as a tumor vascular-targeting agent.

Animals↗

PubMed1998

Tumour radiosensitization by high-oxygen-content gases: influence of the carbon dioxide content of the inspired gas on PO2, microcirculatory function and radiosensitivity.

PURPOSE: To measure the effects of breathing high-oxygen-content gases, with a CO2 fraction of between 0 and 10%, on tumour radiosensitivity, blood flow and oxygenation. METHODS AND MATERIALS: The murine sarcoma F was used, implanted subcutaneously (s.c.) in syngeneic CBA mice. We assessed the induced changes in tumour microregional blood flow and oxygenation using laser Doppler flowmetry, and pO2 histography, respectively. Radiation response was determined using an in vivo-in vitro clonogenic assay 18-20 h post treatment. RESULTS: The results show that the level of radiosensitization achieved is dependent on both the CO2 content of the inspired gas and the duration of gas breathing. No radiosensitization was evident following inhalation of 90% O2 + 10% CO2. All other gases elicited radiosensitization; however, that achieved with 100% O2 disappeared at the extended preirradiation breathing time of 45 min. At this time, radiosensitization was maintained for gases containing 1%, 2.5%, or 5% CO2. Changes in oxygenation, as measured by PO2 electrodes, did indicate improved oxygenation status during inhalation of the gases. However, the time-course and extent of the changes did not mirror accurately the changes in radiosensitization. All the gases with a CO2 content of 2.5% or greater induced a 10-20% reduction in microregional blood flow, with no change evident following inhalation of 100% O2 or 99% O2 + 1% CO2. CONCLUSIONS: The data imply that the decreased radiosensitization seen at extended breathing times of oxygen is unrelated to blood flow changes. The fact that radiosensitization is seen with extended breathing times of gases containing 2.5% and 5% CO2, despite blood flow decreases, is indicative of other overriding physiological changes, perhaps related to oxygen utilisation. The studies overall indicate that, at least in the tumour investigated, radiosensitization is not affected if the CO2 content of the inspired gas is reduced from 5% to 2.5%, or even 1%. Further evaluation of the radiosensitizing effects of such gas mixtures is now warranted. In addition, comparison with recent studies of other tumour types, where carbogen has been shown to improve tumour blood flow, suggests that this may be a tumour-specific phenomenon. Based on these data, further effort is required to elucidate the physiological mechanisms that determine these blood flow changes.

Administration, Inhalation↗