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Clinical Applications. (clinical + applications)
Selected AbstractsGuidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumorsCYTOMETRY, Issue 1 2006Stanley Shackney Abstract Background: Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. Methods and results: We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Conclusions: Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. © 2005 Wiley-Liss, Inc. [source] Efficient hepatocyte engraftment and long-term transgene expression after reversible portal embolization in nonhuman primates,HEPATOLOGY, Issue 3 2009Ibrahim Dagher The feasibility of ex vivo gene therapy as an alternative to liver transplantation for the treatment of liver metabolic diseases needs to be analyzed in large animal models. This approach requires appropriate gene transfer vectors and effective hepatocyte engraftment. Lentiviral vectors have the ability to transduce nondividing differentiated cells, such as hepatocytes, and portal vein occlusion increases hepatocyte engraftment. We investigated whether reversible portal vein embolization combined with ex vivo lentivirus-mediated gene transfer is an effective approach for successful hepatocyte engraftment in nonhuman primates and whether the transgene remains expressed in the long term in transplanted hepatocytes in situ. Simian hepatocytes were isolated after left lobe resection, and the left and right anterior portal branches of animals were embolized with absorbable material. Isolated hepatocytes were labeled with Hoechst dye or transduced in suspension with lentiviruses expressing green fluorescent protein under the control of the human apolipoprotein A-II promoter and transplanted via the inferior mesenteric vein. The whole procedure was well tolerated. The embolized liver was revascularized within 2 weeks. The volume of nonembolized liver increased from 38.7% ± 0.8% before embolization to 55.9% ± 1% after embolization and hepatocytes significantly proliferated (10.5% ± 0.4% on day 3 after embolization). Liver repopulation after transplantation with Hoechst-labeled hepatocytes was 7.4% ± 1.2%. Liver repopulation was 2.1% ± 0.2% with transduced hepatocytes, a proportion similar to that obtained with Hoechst-labeled cells, given that the mean transduction efficacy of simian hepatocyte population was 34%. Transgene expression persisted at 16 weeks after transplantation. Conclusion: We have developed a new approach to improve hepatocyte engraftment and to express a transgene in the long term in nonhuman primates. This strategy could be suitable for clinical applications. (HEPATOLOGY 2009.) [source] Chitosan scaffolds for in vitro buffalo embryonic stem-like cell culture: An approach to tissue engineeringJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2007Wah W. Thein-Han Abstract Three-dimensional (3D) porous chitosan scaffolds are attractive candidates for tissue engineering applications. Chitosan scaffolds of 70, 88, and 95% degree of deacetylation (% DD) with the same molecular weight were developed and their properties with buffalo embryonic stem-like (ES-like) cells were investigated in vitro. Scaffolds were fabricated by freezing and lyophilization. They showed open pore structure with interconnecting pores under scanning electron microscopy (SEM). Higher % DD chitosan scaffolds had greater mechanical strength, slower degradation rate, lower water uptake ability, but similar water retention ability, when compared to lower % DD chitosan. As a strategy to tissue engineering, buffalo ES-like cells were cultured on scaffolds for 28 days. It appeared that chitosan was cytocompatible and cells proliferated well on 88 and 95% DD scaffolds. In addition, the buffalo ES-like cells maintained their pluripotency during the culture period. Furthermore, the SEM and histological study showed that the polygonal buffalo ES-like cells proliferated well and attached to the pores. This study proved that 3D biodegradable highly deacetylated chitosan scaffolds are promising candidates for ES-like cell based tissue engineering and this chitosan scaffold and ES cell based system can be used as in vitro model for subsequent clinical applications. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007 [source] Plaud on Ilardi and FeldmanJOURNAL OF CLINICAL PSYCHOLOGY, Issue 9 2001Joseph J. Plaud A cultural analysis is given to the premise that clinical psychology is a discipline of theoretical fragmentation. It is argued that the discipline will achieve paradigmatic status as an applied science by reestablishing the link between behavioral theory and clinical applications. © 2001 John Wiley & Sons, Inc. J Clin Psychol 57: 1109,1111, 2001. [source] Echogenic liposome compositions for increased retention of ultrasound reflectivity at physiologic temperatureJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2008Kyle D. Buchanan Abstract Targetable echogenic liposomes (ELIP) for ultrasound enhancement of atheroma have recently been developed; however, their retention of echogenicity at physiological temperature is less than desirable. The purpose of this study was to improve ELIP stability and increase clinical potential. The approach utilized the original procedures but involved manipulation of the lipid composition by reducing the level of unsaturation of the phospholipids components to minimize the rate of loss of echogenicity. Echogenicity was measured using a 20 MHz intravascular ultrasound (IVUS) catheter and quantified (as mean gray scale values) using computer-assisted videodensitometry. The optimal preparation for retention of echogenicity stability at physiologic temperature was egg phosphatidylcholine/dipalmitoylphosphatidylcholine/dipalmitoylphos-phatidylethanolamine/dipalmitoylphosphatidylglycerol/cholesterol (27:42:8:8:15, molar percent). This preparation retained 51,±,3.5% of its echogenicity after 1 h at 37°C, more than 5× that retained by the previously descried preparation. In this composition nearly 2/3 of the phosphosphatidylcholine is fully saturated. Such an increase in saturation is anticipated to stiffen the lipid acyl chains. The air pockets that are responsible for reflection of ultrasound waves can be assumed to be stabilized by a lipid monolayer at the interface between the air and bulk water. The increased rigidity of that monolayer is presumed to be responsible for reducing the loss of air and extending the duration of echogenic activity. The stability of this improved formulation now appears to be more than adequate for clinical applications. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2241,2248, 2008 [source] High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolithBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2002Quanzhou Luo Abstract Reactive monoliths of macroporous poly(glycidyl methacrylate- co -ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 481,489, 2002. [source] | ||||||||||