Biomedical subjects

Kevin M Brindle

Publications and source records attributed to Kevin M Brindle.

At least 19 recordsLinked to original sources

PubMed2006

Assessing responses to cancer therapy using molecular imaging.

Tumor responses to therapy in the clinic are still evaluated primarily from non-invasive imaging measurements of reductions in tumor size. This approach, however, lacks sensitivity and can only give a delayed indication of a positive response to treatment. Major advances in our understanding of the molecular mechanisms responsible for cancer, combined with new targeted clinical imaging technologies designed to detect the molecular correlates of disease progression and response to treatment, are set to revolutionize our approach to the detection and treatment of the disease. We describe here the imaging technologies available to image tumor cell proliferation and migration, metabolism, receptor and gene expression, apoptosis and tumor angiogenesis and vascular function, and show how measurements of these parameters can be used to give early indications of positive responses to treatment or to detect drug resistance and/or disease recurrence. Special emphasis has been placed on those applications that are already used in the clinic and those that are likely to translate into clinical application in the near future or whose use in preclinical studies is likely to facilitate translation of new treatments into the clinic.

Antineoplastic Agents↗

PubMed2006

Metabolic consequences of p300 gene deletion in human colon cancer cells.

Metabolite profiling using (1)H nuclear magnetic resonance (NMR) spectroscopy was used to investigate the metabolic changes associated with deletion of the gene for the transcriptional coactivator p300 in the human colon carcinoma cell line HCT116. Multivariate statistical methods were used to distinguish between metabolite patterns that were dependent on cell growth conditions and those that were specifically associated with loss of p300 function. In the absence of serum, wild-type cells showed slower growth, which was accompanied by a marked decrease in phosphocholine concentration, which was not observed in otherwise isogenic cell lines lacking p300. In the presence of serum, several metabolites were identified as being significantly different between the two cell types, including glutamate and glutamine, a nicotinamide-related compound and glycerophosphocholine (GPC). However, in the absence of serum, these metabolites, with the exception of GPC, were not significantly different, leading us to conclude that most of these changes were context dependent. Transcript profiling, using DNA microarrays, showed changes in the levels of transcripts for several enzymes involved in choline metabolism, which might explain the change in GPC concentration. Localized in vivo (1)H NMR measurements on the tumors formed following s.c. implantation of these cells into mice showed an increase in the intensity of the peak from choline-containing compounds in the p300(-) tumors. These data show that NMR-based metabolite profiling has sufficient sensitivity to identify the metabolic consequences of p300 gene deletion in tumor cells in vitro and in vivo.

Animals↗

PubMed2006

Proton NMR analysis of plasma is a weak predictor of coronary artery disease.

Multivariate analysis of 1H-NMR spectra of blood sera was reported previously to predict angiographically defined advanced coronary artery disease (CAD) with >90% accuracy and specificity. The analysis depended mainly on the major lipid regions of the spectra, but many variables, including gender and drug treatment, affect lipid composition and are potential confounders. We have determined the predictive power of the same methodology for angiographically defined CAD using plasma samples from groups of male patients, classified by statin treatment, who had normal coronary arteries (NCAs) or CAD. Predictions for NCA and CAD groups were only 80.3% correct for patients not treated with statins and 61.3% for treated patients, compared with random correct predictions of 50%. A confidence limit of >99% was achieved for 36.2% of predictions for untreated groups and 6.2% for treated groups. Detection of CAD by 1H-NMR with >99% confidence was therefore very weak compared with angiography.

Coronary Artery Disease↗

PubMed2006

Magnetic resonance imaging of functional Schwann cell transplants labelled with magnetic microspheres.

There is increasing interest in the use of magnetic resonance imaging (MRI) methods for tracking the fate of labelled cells in vivo post-implantation. The majority of studies have employed cell labels based on nanometer-sized ultrasmall dextran-coated iron oxide particles (USPIO), which are detected through signal hypointensity in T2-weighted images. Although sensitive to MR detection, these labels can be difficult to distinguish from other sources of signal loss in vivo and can be diluted by cell division. Recently, a micron-sized cell label has been described that is much more sensitive to MR detection and which allows detection of single labels in vivo. We show here that glial cells readily take up this label in culture and that the labelled Schwann cells can be detected in vivo by MRI following their implantation into a demyelinated lesion in the rat spinal cord. Signal loss due to the label is sufficiently great that the labelled cells can easily be distinguished from surrounding haemorrhage at the lesion site. Subsequent histological analysis of the lesion area showed that the transplanted cells were remyelinating the demyelinated axons, demonstrating that the labelled cells retained their biological function and that the majority of the label had remained within the transplanted cells.

Animals↗

PubMed2006

Evaluation of engineered meniscal cartilage constructs based on different scaffold geometries using magnetic resonance imaging and spectroscopy.

Magnetic resonance imaging (MRI) and spectroscopy (MRS) were used to evaluate the properties different scaffold geometries for the production of bioartificial meniscal cartilage constructs. Engineered were generated in perfusion bioreactors from mature sheep meniscal fibrochondrocytes,scaffolds cut from a knitted polyethylene therephtalate (PET) fabric, with a distribution of fibers, 50 microm pores and a density of 45 mg/cm(3) (NF scaffolds), and from two versions of this fabric, which included larger pore sizes (1500 x 500 microm(2)) and densities of (sIV scaffolds) and 83 mg/cm(3) (sV scaffolds). MRI methods were used to determine the permeability the constructs to a low molecular weight MR contrast agent and to measure the macroscopic of medium through and around the constructs. These parameters were correlated with measurements of cell growth and cellular energetics. Cell-free sIV scaffolds were 2- and 5-more porous to flow than the empty sV and NF scaffolds, respectively. These scaffolds, after days of cell growth, were also more permeable to an MR contrast agent. sIV scaffolds yielded(n = 9) with higher cellularities (41 +/- 1%) compared with NF (32 +/- 1%, p < 0.0001) and sV (30 +/- 1%, p < 0.0001) and, when normalized to cell numbers, demonstrated proportionally levels of nucleoside triphosphates (NTP), indicating increased cell viability. Scaffold geometry a marked effect on the properties of engineered meniscal cartilage. MRI and MRS are powerful techniques that can be used to optimize the design of engineered meniscal cartilage and that could be used subsequently to evaluate clinical outcome postimplantation.

Animals↗

PubMed2005

A novel method for observing proteins in vivo using a small fluorescent label and multiphoton imaging.

A novel method for the fluorescence detection of proteins in cells is described in the present study. Proteins are labelled by the selective biosynthetic incorporation of 5-hydroxytryptophan and the label is detected via selective two-photon excitation of the hydroxyindole and detection of its fluorescence emission at 340 nm. The method is demonstrated in this paper with images of a labelled protein in yeast cells.

5-Hydroxytryptophan↗

PubMed2005

Immune cell-induced synthesis of NO and reactive oxygen species in lymphoma cells causes their death by apoptosis.

Induction of apoptosis in a lymphoma cell line using immune cell-conditioned medium, etoposide or an nitric oxide (NO) donor, resulted in the production of reactive oxygen species (ROS). Agents that inhibited NO production or scavenged ROS or species formed by reaction of NO with ROS, protected the cells from apoptosis. These data support the suggestion that immune rejection of an immunogenic derivative of this lymphoma in vivo involves the induced synthesis of both NO and ROS by the tumour cells.

Animals↗

PubMed2005

Discrimination of pathogenic clinical isolates and laboratory strains of Bacillus cereus by NMR-based metabolomic profiling.

Six different Bacillus cereus strains were selected from two different ecotypes: (1) three commonly used laboratory strains that are considered avirulent, and (2) three clinical isolates from meningitis patients. Screening of genomic DNA for the presence of genes encoding known toxins gave no candidate genes that were unambiguously able to distinguish between the two groups. However, the application of multivariate pattern-recognition methods to metabolite profiles derived from the different strains using 1H nuclear magnetic resonance spectroscopy (metabolomics) was able to classify the different profiles. The two different ecotypes were clearly separated on the basis of their metabolite profiles, showing that it is possible to use metabolomic methods to classify pathogens on the basis of their expressed physiology, even when it is not possible to infer a direct mechanistic link to specific virulence factors. This metabolomic approach could also have a wide range of possible applications in both general microbiology and microbial ecology for distinguishing and identifying different functional/physiological ecotypes of bacterial strains or species.

Bacillus cereus↗

PubMed2005

1H MRS-visible lipids accumulate during apoptosis of lymphoma cells in vitro and in vivo.

Proton MRS detection of cellular lipid accumulation has been suggested as a noninvasive method for detecting apoptosis or programmed cell death (PCD) in vivo. The spectral changes that have been observed in apoptotic cells include a general increase in lipid signals and a specific increase in the ratio of the lipid methylene-to-methyl peak intensities. These changes were investigated here following drug-induced apoptosis, both in vitro with a murine lymphoma cell line (EL-4) and in vivo following implantation of these cells to form subcutaneous tumors. Fluorescence microscopy and flow cytometric measurements with a lipophilic dye revealed an accumulation of cytoplasmic lipid droplets in isolated EL-4 cells undergoing etoposide-induced apoptosis. (1)H MR spectra (both diffusion-weighted (DW) and unweighted) showed an increase in lipid signals. However, the methylene/methyl peak ratio showed only minimal changes. Localized in vivo spectroscopy of EL-4 tumors also showed an increase in lipid signals, including a signal from polyunsaturated lipid at 2.8 ppm, after 16-24 h of drug treatment. Again there was no significant change in the methylene/methyl peak ratio. This study confirms that MRS-detectable lipids accumulate in tumor cells undergoing apoptosis, and therefore may be usable as a marker for the noninvasive detection of tumor cell apoptosis in the clinic.

Animals↗

PubMed2005

Monitoring T-lymphocyte trafficking in tumors undergoing immune rejection.

Activated T cells, isolated from animals that had rejected a tumor (E.G7-OVA) expressing chicken ovalbumin, were labeled with citrated superparamagnetic iron oxide nanoparticles at an intracellular iron concentration of up to 0.5 pg/cell. Injection of these labeled T cells into animals bearing E.G7-OVA tumors undergoing immune rejection resulted in tumor infiltration of these cells, which was detectable as a heterogeneous decrease in intensity in T(2)-weighted MR images. T-cell infiltration was confirmed by immunohistochemical staining of tumor sections obtained postmortem and was shown to colocalize with iron that had been stained using Prussian blue. Tumor rejection was correlated with the uptake of labeled T cells, since the infiltration of labeled T cells was only observed in those tumors that went on to regress. This technique should assist in the elucidation of those factors that are important in mediating tumor immune rejection.

Animals↗

PubMed2005

Influence of stirring-induced mixing on cell proliferation and extracellular matrix deposition in meniscal cartilage constructs based on polyethylene terephthalate scaffolds.

The response of engineered meniscal cartilage constructs to stirring-induced mixing in spinner flasks was investigated. Polyethylene terephthalate scaffolds were seeded with meniscal fibrochondrocytes from 6 month-old sheep and cultured under a variety of stirring regimes for 28 days. Stirring-induced mixing increased up to 7-fold the deposition of glucosaminoglycans and up to 3-fold the deposition of collagen, when compared to static cultures. High and medium intensity stirring induced rapid cell proliferation, with maximal cell densities achieved within the first seven days of cultivation. Under these conditions, collagen and glucosaminoglycan deposition occurred predominantly in association with cell proliferation, the specific deposition rate of these biopolymers decreasing markedly after 7 days of cultivation, when the cell number reached a plateau. Constructs exposed to the highest intensity stirring had the highest levels of collagen and glucosaminoglycans and a more homogeneous cell distribution. As the success of the integration at a repair site in the knee of a meniscal construct is likely to be dependent on the cellular activity of the construct, these studies suggest that cultivation of meniscal cartilage constructs, under these conditions, should not extend for more than 7 days.

Animals↗

PubMed2004

Superparamagnetic iron oxide-labeled Schwann cells and olfactory ensheathing cells can be traced in vivo by magnetic resonance imaging and retain functional properties after transplantation into the CNS.

Schwann cell (SC) and olfactory ensheathing cell (OEC) transplantation has been shown experimentally to promote CNS axonal regeneration and remyelination. To advance this technique into a clinical setting it is important to be able to follow the fates of transplanted cells by noninvasive imaging. Previous studies, using complex modification processes to enable uptake of contrast agents, have shown that cells labeled in vitro with paramagnetic contrast agents transplanted into rodent CNS can be visualized using magnetic resonance imaging (MRI). Here we show that SCs and OECs efficiently internalize dextran-coated superparamagnetic iron oxide (SPIO) from the culture medium by fluid phase pinocytosis. After transplantation into focal areas of demyelination in adult rat spinal cord both transplanted SPIO-labeled SCs and OECs produce a signal reduction using T(2)-weighted MRI in anesthetized rats that persists for up to 4 weeks. Although signal reduction was discernable after transplantation of unlabelled cells, this is nevertheless distinguishable from that produced by transplanted labeled cells. The region of signal reduction in SPIO-labeled cell recipients correlates closely with areas of remyelination. Because the retention of functional integrity by labeled cells is paramount, we also show that SPIO-labeled SCs and OECs are able to myelinate normally after transplantation into focal areas of demyelination. These studies demonstrate the feasibility of noninvasive imaging of transplanted SCs and OECs and represent a significant step toward the clinical application of promising experimental approaches.

Animals↗

PubMed2004

Uric acid promotes tumor immune rejection.

Uric acid released from dying cells has been shown recently to act as a danger signal for the immune system, stimulating dendritic cell maturation and enhancing T-cell responses to foreign antigens. Stimulation of dendritic cell maturation by uric acid has been proposed as a mechanism by which the immune system could generate responses against tumors. We show here that uric acid levels are elevated in tumors undergoing immune rejection and that the inhibition of uric acid production, by systemic administration of allopurinol, or the removal of uric acid, by administration of uricase, delayed tumor immune rejection, whereas subcutaneous administration of crystalline uric acid enhanced the rejection process.

Allopurinol↗

PubMed2004

Tumor cell-derived nitric oxide is involved in the immune-rejection of an immunogenic murine lymphoma.

The roles played by host-derived nitric oxide (NO) in the growth and subsequent immune rejection of a immunogenic murine lymphoma were investigated by growing the tumor in mice in which the gene for either inducible NO synthase (iNOS) or endothelial NOS (eNOS) had been ablated. This showed that NO from tumor-infiltrating host cells had no significant effect on either tumor growth or immune rejection, although measurements of tumor nitrite levels and protein nitration showed that there had been significant NO production in the rejected tumors, in both the eNOS and iNOS knockout mice. Inhibition of both tumor and host NOS activities, with an iNOS-selective inhibitor (1400W), a nonselective NOS inhibitor [Nomega-nitro-L-arginine methyl ester (L-NAME)], or scavenging NO with a ruthenium-based scavenger, significantly delayed tumor rejection, while having no appreciable effect on tumor growth. Incubation of tumor cells with medium taken from cultured splenocytes, that had been isolated from immunized animals and activated by incubating them with irradiated tumor cells, resulted in an increase in tumor cell NOS activity and an increase in tumor cell apoptosis, which could be inhibited using L-NAME. We propose that, during the immune rejection of this tumor model, there is induction of tumor NOS activity by cytokines secreted by activated lymphocytes within the tumor and that this results in increased levels of tumor NO that induce tumor cell apoptosis and facilitate immune rejection of the tumor.

Animals↗

PubMed2003

Techniques: Visualizing apoptosis using nuclear magnetic resonance.

Apoptosis plays a key role in tumour biology, and the induction of apoptosis forms a cornerstone of most anticancer therapies. New developments in nuclear magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) have taken these techniques far beyond their original roles as the workhorses of structural and pharmaceutical chemistry and clinical imaging to the detection of previously inaccessible and unrecognized biological phenomena in living cells and tissues undergoing apoptosis. These new MR techniques can be used in the development of new drugs and in the improved detection of treatment responses in the clinic.

Animals↗

PubMed2002

Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria.

Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 microg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200-700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24-84 microg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.

Adipose Tissue, Brown↗

PubMed2002

Differential sensitivity of two adenocarcinoma xenografts to the anti-vascular drugs combretastatin A4 phosphate and 5,6-dimethylxanthenone-4-acetic acid, assessed using MRI and MRS.

The effects of two anti-vascular agents, combretastatin A4 phosphate (CA4P), and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), on the perfusion of two human colon adenocarcinomas implanted in SCID mice, were assessed for up to 3 h using non-invasive magnetic resonance imaging (MRI) and spectroscopy techniques (MRS). MRI measurements of GdDTPA inflow showed that treatment with CA4P had little effect on the perfusion of HT29 tumours. Localized (31)P MRS measurements also showed that the drug had no significant effect on tumour cell energy status, as assessed from the ratio of the integrals of the signals from inorganic phosphate (P(i)) and nucleoside triphosphates. However, after treatment with DMXAA, perfusion was reduced and the P(i)/NTP ratio increased, indicating that the HT29 tumour is susceptible to the action of this drug. The LS174T tumour model was susceptible to both CA4P and DMXAA, using the criteria of changes in GdDTPA inflow and P(i)/NTP ratio.

Adenocarcinoma↗

PubMed2002

Inhibition of proliferative retinopathy by the anti-vascular agent combretastatin-A4.

Retinal neovascularization occurs in a variety of diseases including diabetic retinopathy, the most common cause of blindness in the developed world. There is accordingly considerable incentive to develop drugs that target the aberrant angiogenesis associated with these conditions. Previous studies have shown that a number of anti-angiogenic agents can inhibit retinal neovascularization in a well-characterized murine model of ischemia-induced proliferative retinopathy. Combretastatin-A4 (CA-4) is an anti-vascular tubulin-binding agent currently undergoing clinical evaluation for the treatment of solid tumors. We have recently shown that CA-4 is not tumor-specific but elicits anti-vascular effects in nonneoplastic angiogenic vessels. In this study we have examined the capacity of CA-4 to inhibit retinal neovascularization in vivo. CA-4 caused a dose-dependent inhibition of neovascularization with no apparent side effects. The absence of vascular abnormalities or remnants of disrupted neovessels in retinas of CA-4-treated mice suggests an anti-angiogenic mechanism in this model, in contrast to the anti-vascular effects observed against established tumor vessels. Importantly, histological and immunohistochemical analyses indicated that CA-4 permitted the development of normal retinal vasculature while inhibiting aberrant neovascularization. These data are consistent with CA-4 eliciting tissue-dependent anti-angiogenic effects and suggest that CA-4 has potential in the treatment of nonneoplastic diseases with an angiogenic component.

Angiogenesis Inhibitors↗