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PubMed2002

Lamina propria plasma cells in inflammatory bowel disease: intracellular detection of immunoglobulins using flow cytometry.

This is the first application of flow cytometry for the detection of lamina propria plasma cells and their intracellular immunoglobulins in patients with inflammatory bowel disease compared to healthy controls. The study has been focused on the distribution of IgA, IgG, IgM and the four IgG subclasses. Plasma cells were detected as high CD38 positive cells. For fixation and permeabilisation a single step reagent, Ortho Permeafix, was used. By flow cytometry, in patients with inflammatory bowel disease compared to healthy controls, a higher percentage of IgG+ cells can be observed, in Crohn's disease also a higher percentage of IgM+ cells. Regarding the IgG subclass distribution, patients with Crohn's disease show an increase in IgG2+ cells, patients with ulcerative colitis an increase in IgG1+ and IgG3+ cells. These results do agree with and expand the results of earlier immunohistochemical and functional studies, which are favoured today. For the determination of lymphocyte subset proportions and the detection of intracellular antigens, flow cytometry provides a useful alternative to well-established immunohistochemical methods. By analysing a larger number of cells, this method is more reproducible and less prone to interobserver variations than immunohistochemistry, which needs the pre-selection of a mucosal area, the microscopic scoring of a limited number of cells and the circumvention of high background staining. The optimized flow cytometric protocol used in this study might be a promising tool for further investigations of various purposes.

ADP-ribosyl Cyclase↗
PubMedDate not supplied

Roadmap to a better therapeutic tumor vaccine.

Cell-based cancer vaccines are a highly attractive alternative to standard cancer therapies. They theoretically have the capability of inciting a multitargeted therapeutic response that functions by reshaping the host-tumor interaction, tipping the balance in favor of tumor rejection. Due to the polyclonal immune response induced, they are less likely to result in therapeutic escape than most cancer treatments in use today. Their immune-based mechanism of action offers a unique approach to management that should not be limited by traditional modes of drug resistance. Their favorable side-effect profile further identifies them as a potential treatment modality of choice. Despite these positive features, a number of hurdles must be overcome in order for cancer vaccines to take their place in the clinic as part of standard cancer therapy. Vaccine protocols must be optimized both to induce a high-quality antitumor T-cell response and to abrogate established mechanisms of immune tolerance that actively function to shut antitumor T cells down. By applying basic knowledge of the molecular features of T-cell biology and immune tolerance to the design of trials that combine tumor vaccines with targeted immunomodulatory drugs, potent strategies for inducing effective antitumor immunity can be developed. The first of these combinatorial trials have already been reported and offer a tantalizing glimpse of the future of cancer immunotherapy.

Antigen Presentation↗
PubMed2000

DNA damage induced by radiation of different linear energy transfer: initial fragmentation.

PURPOSE: To investigate DNA fragmentation as a function of linear energy transfer (LET) after exposure to accelerated ions in the LET range 40-225 keV/microm. MATERIALS AND METHODS: Fragmentation patterns of double-stranded DNA in the range 5 kilobasepairs (kbp) to 5.7 megabasepairs (Mbp) were analysed after irradiation of low-passage GM 5758 normal human fibroblast cells with 60Co-photons, helium ions at 40 keV/microm and high-LET nitrogen ions between 80 and 225 keV/microm. Two separate pulsed-field gel electrophoresis protocols were used, optimized for separation of 1-6 Mbp and 5 kbp to 1.5 Mbp fragments. RESULTS: An increased probability of formation of short and medium-sized DNA fragments was revealed following high-LET irradiation. The DNA double-strand break (dsb) induction yields were, respectively, 5.8 and 6.9-8.8 x 10(-9) dsb bp(-1) Gy(-1) for 60Co-photons and ions. The ion yields were some 80-110% higher than those calculated according to a conventional approach, disregarding the fragment distributions. For photons, the yield was 13% higher. The corresponding relative biological effectiveness (RBE) of dsb induction was in the range 1.2-1.5. CONCLUSIONS: A significant non-random contribution to the number of dsb after irradiation with high-LET was confirmed by detailed fragment analysis using pulsed-field gel electrophoresis. The LET had a strong influence on the initial DNA fragment distribution, and hence also on the induction yields measured. However, when the LET was increased to the highest values studied for nitrogen ions, the yield decreased slightly.

Cells, Cultured↗
PubMed1997

An investigation into the effect of input function shape and image acquisition interval on estimates of washin for dynamic cardiac SPECT.

Dynamic cardiac SPECT and PET can be used to measure myocardial perfusion by estimating the kinetic rate constant describing the washing of radioactive-labelled tracers from the blood to the extravascular myocardial tissue. Because of differences in photon statistics and data acquisition techniques, protocols which produce optimal estimates of the washin for dynamic cardiac PET may give suboptimal estimates if applied in dynamic cardiac SPECT. Two important factors in the estimation of washin are the shape of the tracer input function and the image acquisition interval. This study uses computer simulations to investigate the effect of varying the tracer infusion length and image acquisition interval on the bias and variance of estimates of washin obtained with dynamic cardiac SPECT and 99mTc-labelled teboroxime. Bias in parameter estimates can be introduced by aliasing, integration of the time-varying radioactivity by the detector, and detector motion. This bias can be reduced by decreasing the acquisition interval and using a longer-duration input function. However, this results in poor photon statistics, which generate large variance, and can also introduce bias in the estimates of the washin. Our studies indicate that better estimates of the washin are obtained by using an acquisition interval that is of sufficient duration to obtain adequate photon statistics even if this is at the expense of temporal resolution. The increase in bias caused by using a 10 or 20 s acquisition interval instead of a 5 s acquisition interval is minimal when compared with the reduction in variance. Variance in estimates is also reduced by using a sharp input function, resulting in higher peak counts during washin. It is also shown that the variance of estimates of the washin increases generally when faster kinetics are observed. This variance can, however, be reduced by using longer acquisition intervals.

Fourier Analysis↗
PubMed2004

GATE: a simulation toolkit for PET and SPECT.

Monte Carlo simulation is an essential tool in emission tomography that can assist in the design of new medical imaging devices, the optimization of acquisition protocols and the development or assessment of image reconstruction algorithms and correction techniques. GATE, the Geant4 Application for Tomographic Emission, encapsulates the Geant4 libraries to achieve a modular, versatile, scripted simulation toolkit adapted to the field of nuclear medicine. In particular, GATE allows the description of time-dependent phenomena such as source or detector movement, and source decay kinetics. This feature makes it possible to simulate time curves under realistic acquisition conditions and to test dynamic reconstruction algorithms. This paper gives a detailed description of the design and development of GATE by the OpenGATE collaboration, whose continuing objective is to improve, document and validate GATE by simulating commercially available imaging systems for PET and SPECT. Large effort is also invested in the ability and the flexibility to model novel detection systems or systems still under design. A public release of GATE licensed under the GNU Lesser General Public License can be downloaded at http:/www-lphe.epfl.ch/GATE/. Two benchmarks developed for PET and SPECT to test the installation of GATE and to serve as a tutorial for the users are presented. Extensive validation of the GATE simulation platform has been started, comparing simulations and measurements on commercially available acquisition systems. References to those results are listed. The future prospects towards the gridification of GATE and its extension to other domains such as dosimetry are also discussed.

Computer Simulation↗
PubMed2004

Normalized glandular dose (DgN) coefficients for flat-panel CT breast imaging.

The development of new digital mammography techniques such as dual-energy imaging, tomosynthesis and CT breast imaging will require investigation of optimal camera design parameters and optimal imaging acquisition parameters. In optimizing these acquisition protocols and imaging systems it is important to have knowledge of the radiation dose to the breast. This study presents a methodology for estimating the normalized glandular dose to the uncompressed breast using the geometry proposed for flat-panel CT breast imaging. The simulation uses the GEANT 3 Monte Carlo code to model x-ray transport and absorption within the breast phantom. The Monte Carlo software was validated for breast dosimetry by comparing results of the normalized glandular dose (DgN) values of the compressed breast to those reported in the literature. The normalized glandular dose was then estimated for a range of breast diameters from 10 cm to 18 cm using an uncompressed breast model with a homogeneous composition of adipose and glandular tissue, and for monoenergetic x-rays from 10 keV to 120 keV. These data were fit providing expressions for the normalized glandular dose. Using these expressions for the DgN coefficients and input variables such as the diameter, height and composition of the breast phantom, the mean glandular dose for any spectra can be estimated. A computer program to provide normalized glandular dose values has been made available online. In addition, figures displaying energy deposition maps are presented to better understand the spatial distribution of dose in CT breast imaging.

Body Burden↗
PubMed2003

Perturbation of blood flow as a mechanism of anti-tumour action of direct current electrotherapy.

Anti-tumour effects of direct current electrotherapy are attributed to different mechanisms depending on the electrode configuration and on the parameters of electric current. The effects mostly arise from the electrochemical products of electrolysis. Direct toxicity of these products to tumour tissue is, however, not a plausible explanation for the observed tumour growth retardation in the case when the electrodes are placed into healthy tissue surrounding the tumour and not into the tumour itself. The hypothesis that the anti-tumour effectiveness of electrotherapy could result from disturbed blood flow in tumours was tested by the measurement of changes in blood perfusion and oxygenation in tumours with three different methods (in vivo tissue staining with Patent Blue Violet dye, polarographic oximetry, near-infrared spectroscopy). The effects induced by electrotherapy were evaluated in two experimental tumour models: Sa-1 fibrosarcoma in A/J mice and LPB fibrosarcoma in C57B1/6 mice. We found that perfusion and oxygenation were significantly decreased after electrotherapy. Good agreement between the results of different methods was observed. The effect of electrotherapy on local perfusion of tumours is probably the prevalent mechanism of anti-tumour action for the particular type of electrotherapy used in the study. The importance of this effect should be considered for the optimization of electrotherapy protocols in experimental and clinical trials. The non-invasive technique of near-infrared spectroscopy proved to be a reliable method for detecting perfusion and oxygenation changes in small solid tumours.

Animals↗
PubMed2002

Rapid screening for high-titer retroviral packaging cell lines using an in situ fluorescence assay.

The production of high-titer recombinant retrovirus is a major determinant of the efficiency of target cell transduction. Titer assessment for producer clones that contain vectors encoding proteins that can be detected using fluorescence is typically performed by flow cytometry. However, this method is both costly and labor intensive, severely limiting the number of clones that can be screened for each construct. In this report we describe a rapid, high-throughput screening method for viral quantitation of producer clone supernatant on target cells using a 96-well format. Plates were assayed using a multichannel fluorescent reader to determine the percentage of target cells expressing green (EGFP), cyan (ECFP), yellow (EYFP) or red (DsRed) fluorescent reporter genes, or their combinations. The relative fluorescence counts of target cells incubated with viral supernatant from each packaging cell clone correlated with the level of transduction, and hence, viral titer. Correlation of cell fluorescence between the fluorescent plate reader assay and flow cytometric assessment was high (r(2) = 0.96). Independent detection of different fluorescent reporters enabled multiplex assays to be performed. Simultaneous cell density analysis using alamarBlue fluorescence was proportional to cell number per well (r(2) = 1.0). In situ titer assessment of 66 FLYRD packaging cells encoding the EGFP reporter gene identified clones (>10(7) colony forming units per milliliter [CFU/ml]) that provided titers up to sevenfold over the parent population. The application of this rapid, high-throughput screening method overcomes many limitations imposed by the current flow cytometric screening method. This robust assay maximizes the chance of identifying rare high-titer packaging clones and offers a further opportunity to optimize gene transfer protocols.

Cell Line↗
PubMed2001

Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium.

Cord blood (CB) and fetal liver (FL) cells are two alternative sources of human hematopoietic stem cells. Optimization of cryopreservation protocols is an important aspect in the banking of these tissues. Out of the multiple factors responsible for cryodamage of cells, membrane leakage and oxygen free-radical generation have been shown to contribute substantially toward the process. We have studied the effect of certain additives, like membrane stabilizers and bioantioxidants, to the conventional freezing medium on viability, nucleated cell recovery, and clonogenic potential of frozen cells. Our results show that trehalose, a membrane stabilizer, when used in combination with 10% dimethyl sulfoxide (DMSO) affords better cryoprotection as evidenced by significantly increased colony formation as compared to 10% DMSO alone. The cryoprotection afforded by trehalose persists at least for 1.5 years and there is no bias toward protection of a particular lineage. We also found that this increased cryoprotective effect of trehalose is seen both at -196 degrees C and -80 degrees C storage temperatures. Addition of taurine, an amino acid, another membrane stabilizer, and a natural cryoprotectant to the traditional freezing medium also results in beneficial effect. Of the three bioantioxidants tested, i.e., ascorbic acid, alpha-tocopherol acetate, and catalase, catalase shows maximum cryoprotective effect both at -196 degrees C and at -80 degrees C. Because the mode of cryoprotective action of catalase and trehalose are totally different, we tried a combination of these two compounds along with 10% DMSO. At -196 degrees C the protection afforded by the combination was significantly better than that afforded by individual components. At -80 degrees C, however, the combination did not give any added protection as compared to the individual single additives, although it was significantly better than 10% DMSO alone. These results indicate that the addition of membrane stabilizers and antioxidants to the conventional freezing medium may help to improve post thaw recovery of hematopoietic cells.

Antioxidants↗
PubMed1993

Radiological considerations: percutaneous laser disc decompression.

Diagnostic radiology is an integral part of percutaneous laser disc decompression (PLDD). All physicians involved in PLDD patient selection and treatment must be familiar with the imaging techniques unique to this procedure to ensure a successful outcome. The following review is based on the cumulative experience gained in performing over 150 PLDD procedures. It discusses the function of diagnostic radiology in all facets of PLDD including patient selection, intraoperative imaging, postoperative evaluation, and analysis of complications. Fundamental radiologic concepts that apply to PLDD are explained and protocols suggested to optimize results and avoid complications.

Humans↗
PubMed1995

The role of radiology in percutaneous laser disc decompression.

Diagnostic imaging has always played a key role in the evaluation of patients with suspected lumbar disc herniation. With the development of percutaneous laser disc decompression (PLDD), radiology is now equally important in its treatment. All physicians involved with the PLDD candidate must be familiar with the imaging techniques unique to this procedure to ensure a successful outcome. The following review is based on the cumulative experience gained in performing over 200 PLDD procedures. It discusses the function of diagnostic radiology in all facets of PLDD including patient selection, intraoperative imaging, postoperative evaluation, and analysis of complications. Fundamental radiologic concepts that apply to PLDD are explained and protocols suggested to optimize results and avoid complications.

Diskectomy, Percutaneous↗
PubMed1998

Correction of respiratory burst activity in X-linked chronic granulomatous cells to therapeutically relevant levels after gene transfer into bone marrow CD34+ cells.

Chronic granulomatous disease (CGD) is a disorder of the lymphohematopoietic system, whereby phagocytes of affected patients are unable to kill microorganisms. CGD is caused by a functional defect in the phagocytic nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase (phox) enzyme complex, leading to a lack of microbicidal metabolites. As a therapeutic approach toward the predominant X-linked form of CGD, we have developed a bicistronic retroviral vector containing the coding sequences of gp91-phox and a cytoplasmically truncated version of the human low-affinity receptor for nerve growth factor (deltaLNGFR). Full reconstitution of superoxide-generating activity was achieved with this vector in a gp91-phox-deficient cell line. Using an optimized gene transfer protocol, up to 85% of the CD34+ cells obtained from the bone marrow of X-CGD patients were transduced. CD15+ cells differentiated in vitro from transduced X-CGD CD34+ cells showed correction of NADPH oxidase activity to 45-52% of normal levels whereas deltaLNGFR expression was found in 40-67% of the CD15+ cells. Moreover, immunoblots prepared from extracts of transduced CD15+ cells revealed gp91-phox protein levels similar to those found in neutrophils derived from normal CD34+ cells. Taking into consideration that superoxide production in only 5 to 10% of wild-type neutrophils is sufficient to protect X-CGD heterozygotes from serious infections, the results achieved in this study shows that for X-CGD patients a curative approach based on the genetic modification of hematopoietic stem/progenitor cells is feasible.

Antigens, CD34↗
PubMed1987

Medical monitoring. What is it, how can it be improved?

Little has been published about optimizing medical monitoring protocols, although monitoring accounts for over half of all medical laboratory tests. Monitoring is a form of surveillance consisting of repeated testing intended to detect a specified change in a patient indicating a change in his prognosis, need for treatment or need for a change in treatment. The concept of monitoring overlaps with those of screening and diagnosis. One may monitor a physiologic or pathologic process, a therapeutic or noxious agent, comparing results with the patient's previous results or with a group reference range. Pertinent questions include: what are the indications, what is to be monitored, which tests are to be used, when to start, how frequently to test, when to stop. Preliminary improvement of monitoring protocols may be achieved by applying common sense guidelines for medical decision making; using best estimates of test characteristics, of possible risks and benefits of testing, not testing, treating and not treating. Sensitivity analysis may help determine when further clinical research is needed.

Decision Making↗
PubMed1994

Simultaneous detection of X-chromosome loss and non-disjunction in cytokinesis-blocked human lymphocytes by in situ hybridization with a centromeric DNA probe; implications for the human lymphocyte in vitro micronucleus assay using cytochalasin B.

A methodology for the simultaneous detection of chromosome loss and gain in mammalian cells has been developed which is based upon the analysis of chromosome distribution in daughter nuclei of binucleated human lymphocytes. X-chromosome distribution was followed by in situ hybridization, using a commercial biotinylated DNA probe specific for the centromeric alphoid sequences of human X-chromosome. In order to optimize the experimental protocol for the use of cytokinesis-blocked lymphocytes in aneuploidy assays, the effect of harvest time and cytochalasin B (Cyt B) dosage upon chromosome distribution was investigated. To this end, lymphocyte cultures were treated 44 h after mitogen stimulation with different dosages of Cyt B and collected at 60, 66 and 72 h. High rates of binucleated cells with unbalanced chromosome distribution (two spots in one nucleus and none in the other in male cells; three spots in one nucleus and one in the other in female cells) and abnormal spot number (more than or less than two per male cell or four per female cell) were observed at 66 and 72 h in cultures treated with the lowest Cyt B dose (3 micrograms/ml). In contrast, low frequencies of unbalanced or abnormal binucleated cells were observed at 60 h with both 3 and 6 micrograms/ml Cyt B. These results indicate that binucleated lymphocytes with low background frequencies of malsegregation (required for the analysis of induced aneuploidy), can be obtained by harvesting lymphocyte cultures 60 h after stimulation (16 h after Cyt B block).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1998

Preselection of shotgun clones by oligonucleotide fingerprinting: an efficient and high throughput strategy to reduce redundancy in large-scale sequencing projects.

Large-scale genomic sequencing projects generally rely on random sequencing of shotgun clones, followed by different gap closing strategies. To reduce the overall effort and cost of those projects and to accelerate the sequencing throughput, we have developed an efficient, high throughput oligonucleotide fingerprinting protocol to select optimal shotgun clone sets prior to sequencing. Both computer simulations and experimental results, obtained from five PAC-derived shotgun libraries spanning 535 kb of the 17p11.2 region of the human genome, demonstrate that at least a 2-fold reduction in the number of sequence reads required to sequence an individual genomic clone (cosmid, PAC, etc.) can be achieved. Treatment of clone contigs with significant clone overlaps will allow an even greater reduction.

Chromosomes, Human, Pair 17↗
PubMed1999

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.

We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).

Cloning, Molecular↗
PubMed2001

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.

Fabaceae↗
PubMed2004

What is the best way to assess microsatellite instability status in colorectal cancer? Study on a population base of 462 colorectal cancers.

The assessment of the microsatellite instability (MSI) status in colorectal cancers is presently warranted for three reasons: 1) as a screening tool for hereditary nonpolyposis colorectal cancer, 2) as a prognostic marker, and 3) as a potential predictive factor of chemotherapy response. The aim of this study was to evaluate, on a large scale with tissue samples coming from a number of different sources, the difficulties met with routine use of immunohistochemistry (IHC) and to determine if it really does offer an accurate alternative to PCR genotyping. Colorectal carcinomas from 462 consecutive patients resected in public or private hospitals were assessed for MSI status by two methods: MSI testing (with BAT-26 microsatellite) and IHC detection of hMLH1, hMSH2, and hMSH6 proteins. Of the 398 cancers tested, immunohistochemistry was noncontributory in 42 (10.5%), focal in 9 (2.3%), and discordant with the PCR results in 36 (9%). For these 87 cases, complementary analyses were performed to explain discrepancy. After additional IHC assay with modified processing protocols, 8 cases remained noncontributory, 2 focal, and 28 discordant: 18 microsatellite stability IHC/MSI PCR and 10 MSI IHC/microsatellite stability PCR. For these discordant cases, we performed a multiplex PCR assay on DNA extracted from the frozen sample and BAT-26 was amplified from DNA extracted from the paraffin blocks used for IHC. Four discordant cases were reclassified after PCR multiplex assay (3 as MSI and 1 as microsatellite stability). Five other cases displayed intratumoral heterogeneity and 19 remained discordant. The discrepancy could be partly explained by variable technical protocols of fixation in the different laboratories, leading to variations in staining quality and difficulties in IHC interpretation. This population-based study is the first one to show that IHC is not sensitive and specific enough to be used routinely. Immunohistochemistry analysis of MMR proteins must be performed in standardized conditions and interpreted by confirmed pathologists. It cannot replace PCR as long as protocols are not optimized and harmonized.

Aged↗