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In cancer and AIDS, overexpression of the MDR1 gene has important implications in the design of chemotherapy protocols because of the ability of its product, the ATPdependent drug efflux pump P-glycoprotein (Pgp), to confer selective advantage to tumor and HIV-infected cells in the form of multidrug resistance. To study Pgp expression and physiology, we designed a translational fusion between the MDR1 and enhanced green fluorescent protein (EGFP) genes. The chimeric protein, Pgp-EGFP, was concentrated mainly in the plasma membrane and in the Golgi when expressed in drug-sensitive KB- 3-1 cells. Doxorubicin, daunorubicin and rhodamine-123 efflux assays confirmed function of the chimeric pump. Also, at the single-cell level, an inverse relationship between Pgp-EGFP expression and nuclear doxorubicin accumulation was demonstrated. Polarized Pgp expression on the apical cell surface was confirmed by transfection of the MDR-EGFP fusion gene into MDCK cells. However, after colchicine selection, Pgp-EGFP was also detectable in the lateral domain of the transfected MDCK monolayers. These results indicate that drug selection affects not only expression, but cellular localization of Pgp. Furthermore, using a tet-based inducible expression system for Pgp-EGFP, we confirmed the stable nature of Pgp (t1/2 total Pgp-EGFP= 2.2 days), but revealed that surface-Pgp acquires extra stability as an active pump (t1/2 surface Pgp-EGFP= 3.7 days).

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Servicios de Hemoterapia-Hemostasia, Hospital Clinic, IDIBAPS, Barcelona, Spain. mpino@clinic.ub.es

BACKGROUND: Chronic renal failure patients exhibit accelerated atherosclerosis, which is associated with a high incidence of cardiovascular death. We investigated the potential effect of uraemic medium on cell proliferation and apoptosis of endothelial cells in culture (ECs), two key processes in the development of atherosclerosis. Phosphorylation kinetics of the mitogen-activated protein kinase (MAPK) p42/44 and p38 were also evaluated. METHODS: ECs were cultured with growth media supplemented with pooled sera from healthy donors. Semiconfluent ECs were incubated for 24 h with media supplemented with pools of control or uraemic sera. Cell proliferation was assessed through morphometric analysis and by flow cytometry evaluation of cell cycle. To investigate if uraemic medium induces apoptosis in ECs, we used a combination of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay and activation of caspase-3 using flow cytometry. Changes in the phosphorylation levels of MAPK were evaluated in cell lysates by western blotting. RESULTS: Exposure to uraemic media caused an alteration in the morphology of ECs, showing irregular shape and size. The number of ECs at S+G(2)M phase in the cell cycle was found to be increased when exposed to uraemic media for 24 h (28.4+/-2.9 vs 20.2+/-2.6% in control ECs). There was a transient increase in levels of phosphorylation of MAPK in both cells, although these levels were significantly higher in ECs exposed to uraemic media, especially after 5 min. In contrast, no signs of apoptosis were observed in ECs incubated with uraemic medium at the conditions applied. CONCLUSIONS: Under our experimental conditions, uraemic medium accelerates proliferation of ECs, but it does not seem to induce apoptosis. The increased proliferation observed could be related to a higher MAPK activity in these cells. Although the enhanced atherosclerosis cannot be explained on the basis of an apoptotic process, the proliferative status could contribute to intimal proliferation, which is considered to be an earlier step in the development of atherosclerosis.

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Service of Haematotherapy and Haemostasis, Hospital Clinic and IDIBAPS, Barcelona, Spain.

Thrombotic thrombocytopenic purpura (TTP) is a rare but severe disorder characterized by microangiopathic haemolytic anaemia, consumptive thrombocytopenia, neurological involvement and formation of platelet thrombi in the small vessels. The aetiopathology of TTP and the mechanism behind the beneficial effect of plasma exchange with plasma infusion have not yet been fully elucidated. We have studied the effect of plasma from four patients with TTP on human blood phagocyte activation, as measured by reactive oxygen species (ROS) production and CD11b expression. TTP plasma obtained in the acute phase of the disease induced ROS production by human monocytes and enhanced CD11b expression on neutrophils. This activation effect remained in the cryosupernatant but not in the cryoprecipitate made from TTP plasma, and disappeared when a complete response was achieved by plasma exchange. These findings suggest that activated blood phagocytes may be involved in the pathophysiology of TTP.

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Department of Hematotherapy, Hospital Clinic, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain.

Nucleated red blood cells (NRBCs) are involved in normal physiologic processes, as well as in several malignancies. They are usually counted manually under the microscope. However, blood sample manipulation may be a source of variability and manual counting is imprecise, time-consuming, and subjective. To improve identification of CD45-negative cells, we used a flow cytometry technique that avoids the addition of lysing reagents and stains viable cell nuclei. We applied this method for counting and isolating NRBC subpopulations in whole blood samples, using DNA/RNA viable staining to discriminate nonnucleated erythroid cells and debris. NRBC counts gave 197.95 cells per mm(3) in mobilized peripheral blood samples (1.00%, n = 20), 3897.59 cells per mm(3) in leukapheresis products (3.08%, n = 20), and 765.21 cells per mm(3) in cord blood samples (6.09%, n = 20). Normal bone marrow counts were 5449.42 cells per mm(3) (11.76%, n = 20). Scatter profiles showed three distinct populations, from early to late-stage erythroblasts, consisting of erythroblasts, orthochromatic erythroblasts, and ejected nuclei, as confirmed by Wright-Giemsa staining. In addition, flow cytometry immunophenotyping showed that glycophorin A was expressed dimly on NRBCs during maturation. These findings point to the feasibility of live NRBCs studies, which offer great potential for a wide range of disciplines. Copyright 2002 Wiley-Liss, Inc.

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Servei d'Hemoterapia i Hemostasia, Hospital Clinic, Institut d'Investigacions Biomediques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain.

BACKGROUND: We previously developed a method for counting CD34(+) cells in unlysed whole blood. This method was applied to normal human bone marrow, peripheral blood after mobilization of progenitor cells, leukapheresis products, and cord blood and was validated with two different lyse-no wash methods. However, the main advantage that we described, erythrocyte discrimination using nucleic acid staining, was also the main restriction because additional markers for the immunologic characterization of CD34(+) cells cannot be included.
METHODS: We used SYTO-13 and fluorescein isothiocyanate (FITC)-CD45 staining (FL1) instead of SYTO-13 and phycoerythrin (PE)Cy5-CD45 staining (FL3) to leave the third and fourth fluorescence parameters available for further characterization of CD34(+) cells. The new method was validated by applying it to cord blood samples (n = 20).
RESULTS: FITC-CD45 antibody gave a 1.7-fold increase in mean fluorescence intensity over SYTO-13 alone. From absolute counts (CD34(+) cells per microliter), we plotted the differences between the methods against their mean, showing that differences fell into acceptable ranges.
CONCLUSIONS: No-lyse procedures may represent an advance for cell immunophenotyping and it could be applied to the measurement of additional markers. Cytometry (Clin. Cytometry) 50:249-253, 2002. Copyright 2002 Wiley-Liss, Inc.

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Centre de Genetica Medica i Molecular, Institut de Recerca Oncologica, Hospital Duran i Reynals, 08907 L'Hospitalet de Llobregat, Barcelona, Spain.

BACKGROUND: Chimeraplasty is a novel methodology that uses chimeric RNA/DNA oligonucleotides (chimeraplasts) to stimulate genomic DNA repair. Efficient uptake and nuclear localization of intact chimeraplasts are key parameters to achieve optimal correction of mutation defects into specific cell types.
METHODS: A 5'-end FITC-labeled 68-mer RNA/DNA oligonucleotide was complexed with the polycation polyethylenimine (PEI) and the cationic lipids Cytofectin and GenePorter. Flow cytometry was employed to evaluate chimeraplast uptake under different conditions. Intracellular chimeraplast distribution and co-localization with endocytosis markers were assessed by confocal microscopy. Relative quantification of chimeraplast metabolism was performed by denaturing PAGE and GeneScan(trade mark) analysis.
RESULTS: In airway epithelial cells, optimized chimeraplast uptake reached near 100% efficiency with the carriers tested. However, chimeraplast nuclear localization could only be achieved using PEI or Cytofectin. Chimeraplast/GenePorter lipoplexes were retained in the cytoplasm. PEI polyplexes and Cytofectin lipoplexes displayed different uptake rates and internalization mechanisms. Chimeraplast/PEI polyplexes were internalized at least partially by fluid-phase endocytosis. In contrast, phagocytosis may have contributed to the internalization process of large-sized chimeraplast/Cytofectin lipoplexes. Moreover, significant chimeraplast degradation was detected 24 h after transfection with both PEI polyplexes and Cytofectin lipoplexes, although the latter seemed to confer a higher degree of protection against nuclease degradation.
CONCLUSION: Both Cytofectin and PEI are efficient for chimeraplast nuclear uptake into airway epithelial cells. However, despite the distinct structures and trafficking pathways of the corresponding complexes, none of them could prevent nuclease-mediated metabolism of the chimeric oligonucleotides. These findings should be taken into account for future investigations of chimeraplast-mediated gene repair in airway epithelial cells. Copyright 2002 John Wiley & Sons, Ltd.

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Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Barcelona, Marti i Franques, Spain. domingo@sun.bq.ub.es

Immunomagnetic systems have been used for positive selection of a cell fraction from a mixture using appropriate surface markers with satisfactory results, as haematopoietic CD34+ cells. This work reports on the development of poly(ethylene glycol)-grafted (PEG) immunoliposomes loaded with citrate-magnetite stabilized particles as the separation vehicles for immunomagnetic separations. The magnetic ferrofluid was encapsulated into PEG-liposomes by the DRV methodology. The magnetoliposomes had a liposomal size of approximately 450 nm and a Fe/lipid molar ratio of 1.52+/-0.26, and were retained in the magnetic field created by the MiniMACS system. Anti-CD34 immunomagnetoliposomes with 100 mAb/vesicle were prepared by coupling the My10 mAb and bound specifically for CD34+ KG-1a cells in culture and in mixtures with CD34-cells (CHO or Jurkat). The magnetic cell sorting was carried out in cell mixtures KG-1a/CHO or KG-1a/Jurkat with different initial% of CD34+ Kg-1a cells. For 10(6) positive cells and 100 microM of immunomagnetoliposomes, the capture efficiency was > 85% and independent of the starting percentage of CD34+ cells. The decrease of the final purity, when the starting percentage of CD34+ cells decreases and, dependent of the CD34- cell line used, point to the degree of non-specific cell binding of My10-immunomagnetoliposomes as being crucial, among of the methodological aspects as the number of starting CD34+ cells. The CD34+ cells isolated retained the viability with an estimated recovery of 45-50%.

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Editorial

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Institut de Recerca Oncologica, Hospital Duran i Reynals, L'Hospitalet, Barcelona, Spain.

The aberrant content of DNA, or aneuploidy, is a hallmark of tumor cells and may be associated with malignant potential. Based on the hypothesis that aneuploidy, as a form of genetic instability, results in an increased capability to generate cell heterogeneity, we investigated whether a comprehensive assessment of aneuploidy extent and degree might be a reliable indicator of tumor aggressiveness. DNA content was determined by flow cytometry in the infiltrating front of 131 paraffin-embedded primary colorectal carcinomas collected in a prospective design. Enrichment of tumor cells by sample microdissection resulted in neoplastic cell contents above 75%. An estimate of aneuploidy, the aneuploidy index (AI), was calculated as the tumor DNA content adjusted by the percentage of diploid and aneuploid cells in G0/G1. Thirty-nine tumors were diploid, 90 hyperdiploid, and 2 hypodiploid. The mean AI in aneuploid tumors was 1.20+/-0.17 and correlated with Dukes' stage and metastasis (p < 0.05). A high AI (receiver operating characteristic curve cutoff value greater than 1.14) predicted a poorer outcome in univariate (p = 0.004) and multivariate (p = 0.01) analyses. Based on these results, we postulate that aneuploidy is the molecular engine of progression in a subset of colorectal cancers, in which the AI seems to be a sensible and independent gauge of malignant potential. The AI determination may have prognostic application in colorectal cancer, especially in low-grade tumors, which might benefit from coadjuvant therapies.

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Department of Cryobiology and Cell Therapy, Cancer Research Institute, Gran Via km 2.7, 08907 Barcelona, Spain.

Hematopoietic stem cells are able to self-renew and to differentiate into progenitors of all hematopoietic lineages; these processes are regulated by many cytokines. A subset of hematopoietic cells, presumably containing hematopoietic stem cells, expresses the cell-surface antigen CD34. CD34+-purified fractions are enriched in colony-forming units and long-term culture initiating cells, whereas CD34- fractions are depleted. CD34+ cells are commonly used in hematopoietic stem cell transplantation. They can be mobilized from bone marrow into peripheral blood by means of chemotherapy and/or cytokine stimulatory treatments, and then collected for use in cancer therapy, hematopoietic stem cell expansion studies and gene therapy. The widening use of stem cell mobilization means that a method that accurately and rapidly quantifies CD34+ cells is very much needed. Although several methods for CD34+ quantification have been proposed, there is a lack of consensus on a method to count this rare population.

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Department of Biochemistry and Molecular Biology, University of Barcelona, Spain.

Several methods for the preparation of sterically stabilized immunoliposomes (SIL) have recently been described. This report examines an established method for coupling anti-CD34 My10 mAb to poly(ethylene glycol)-liposomes (PEG-liposomes) containing the anchor pyridyldithiopropionylamino-PEG-phosphatidylethanolamine (PDP-PEG-PE) via a cleavable disulfide bond. Efficient attachment of pyridyldithio-derivatized mAb took place (equivalent to coupling ca. 70% of total input protein) at 2 mol percent of the functionalized PEG-lipid. The My10-SIL bound specifically to CD34+ cells (human leukemic KG-1a and hematopoietic progenitor cells) and the extent of binding was a function of liposomal lipid concentration, the mAb density in the liposome surface and the CD34 cell expression. In mixtures with CD34- cells (CHO or Jurkat), CD34+KG-1a cells were determined by flow cytometry at percentages (1-4%) similar to those reported in clinical samples (such as cord blood, mobilized peripheral blood and bone marrow) using a direct immunostaining with My10-SIL. The disulfide bond was stable in cell culture medium (10% of fetal calf serum) during 8 h and cell-bound SIL can be released from cells by treatment with dithiothreitol as reducing agent under mild conditions (1 h of incubation with 50 mM DTT at 20 degrees C). SIL binding and subsequent dithiothreitol treatment did not influence the cell viability. Our approach should contribute to the development of targetable liposomal vehicles to CD34+ cells for use in ex vivo conditions as sorting of hematopoietic stem cells.

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Department of Cryobiology and Cell Therapy, Institut de Recerca Oncologica, L'Hospitalet, Barcelona, Spain.

BACKGROUND AND OBJECTIVE: Successful gene therapy applications require optimized strategies to increase gene transfer efficiency into hematopietic progenitor cells (HPCs) with long-term repopulating ability. One of the issues that needs to be clarified is how hematopoietic cells proliferate, differentiate and express the transgene after each cycle of transduction. We investigated the kinetics of cell expansion, CD34 antigen expression and transduction efficiency of human hematopoietic cells in culture conditions commonly used in retroviral gene transfer protocols.
DESIGN AND METHODS: Purified CD34+ cells from cord blood (n=5) or leukapheresis products (n=9) and a retroviral vector encoding an enhanced version of the green fluorescent protein (EGFP) were used. Target cells were exposed daily to vector-containing supernatants and a combination of interleukin 3 (IL-3), interleukin 6 (IL-6), stem cell factor (SCF) and Flt3-ligand (FL). Cell samples were harvested from the cultures and analyzed at 24 hour intervals for seven consecutive days.
RESULTS: We found that CD34+ cells proliferated and differentiated under our culture conditions. The number of genetically modified cells increased after each cycle of transduction. Median numbers of cells positive for both CD34 and EGFP increased steadily over the culture period, but after day four most of the EGFP+ cells had a low CD34 expression.
INTERPRETATION AND CONCLUSIONS: Culturing and transducing CD34+ cells for longer periods of time under these conditions might be detrimental for ex vivo gene transfer applications since the transduced cells are likely to have a decreased potential for long-term engraftment and repopulation in vivo.

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Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Barcelona, Marti i Franques, 1, 08028, Barcelona, Spain.

Several methodologies for the preparation of polyethylene glycol-grafted immunoliposomes have been developed by attaching antibodies to the terminus of the polymer. Unilamellar liposomes were prepared containing a combination of a functionalized polyethylene glycol(3400) and an inert polyethylene glycol(2000) phosphatidylethanolamine derivate up to 5 mol%. The greater length of the functionalized polyethylene glycol derivate did not alter the liposomal sterical stability or the remote loading of doxorubicin. Anti-CD34 immunoliposomes were prepared by the reaction of maleimide-derivatized My10 antibody with generated thiol groups at the periphery of the liposomes and efficiencies of nearly 100% were obtained. The greater accessibility of the reactive group makes this strategy more efficient than others described. The immunoliposomes prepared bound specifically to CD34+ cells.

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Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Barcelona, Marti i Franques, 1, 08028 Barcelona, Spain.

The My-10 monoclonal antibody has facilitated the search of haematopoietic stem cells by recognizing selectively the human CD34 antigen. In the present work, My-10 immunoliposomes directed specifically against CD34+ cells were prepared, characterized and tested in vitro. Binding to target cells at 4 degreesC of immunoliposomes containing carboxyfluorescein as aqueous marker was evaluated by flow cytometry and fluorescence microscopy. These immunoliposomes demonstrated their capacity to bind specifically to CD34+ cells. Studies have shown that 9 antibodies/vesicle were sufficient to obtain a good binding efficiency. The product was stable over one month at 4 degreesC in terms of leakage of encapsulated carboxyfluorescein, particle size and antigen binding capacity. Copyright 1998 Elsevier Science B.V.

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Departament de Criobiologia i Terapia Cellular, Institut de Recerca Oncologica, Hospital Duran i Reynals, Barcelona, Spain.

The product of the mdr1 gene, P-glycoprotein (P-gp), represents a common mechanism of cellular resistance to a wide variety of structurally and functionally unrelated drugs. A range of structurally different P-gp inhibitors, such as verapamil, cyclosporin A and SDZ PSC 833, have been shown to modify multidrug resistance (MDR). We used flow cytometry to investigate in vitro modulation of P-gp-dependent efflux of rhodamine 123 (Rh123). The capacity to modulate the MDR phenotype of vinblastine-resistant Chinese hamster ovary (CHO) cells was assessed by analyzing the concentration of modulator needed to decrease the Rh123 mean fluorescence intensity by 50%. We found that the cyclosporin derivative SDZ PSC 833 was significantly more effective than cyclosporin A and verapamil, either in the presence or absence of fetal calf serum-supplemented media. This study indicates that analysis of Rh123 efflux modulation can be used to determine the optimal doses of MDR inhibitors in vitro and suggests that more than one modulator is needed to measure P-gp function, since verapamil had no effect on Rh123 modulation when MDR cells were used.

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Departament de Criobiologia i Terapia Cellular, Institut de Recerca Oncologica, Hospital Duran i Reynals, Barcelona, Spain.

The MDR1 gene product, P-glycoprotein (P-gp), works as a transmembrane efflux pump for several cytotoxic products, representing a major cause for cancer treatment failure. Rhodamine 123 (Rh123), a low toxic fluorescent probe commonly used to assess mitochondrial bioenergetics in living cells, has also been used to measure the efflux activity of P-gp in both normal and malignant cells. Analysis of variation in cellular fluorescence by measuring the rates of Rh123 influx and efflux, together with the effect of mdr reversing agents, allows the investigation of drug-resistant phenotypes in cancer samples. We have studied the functional activity of P-gp in human leukemic cell lines using flow cytometry, taking into consideration that variables such as Rh123 cytotoxicity, culture conditions, cell membrane integrity, as well as the effect of specific P-gp modulators, can impair the resolution of the Rh123-efflux measurements. The studies show that: (1)optimal non-cytotoxic concentrations of Rh123 which allow appropriate color compensation are in the range of 50-200 ng/ml; (2) life-gating allows accurate measurement on the 50% average rate of Rh123 efflux; (3) relative efficiency of P-gp inhibitors was PSC-833 > cyclosporin A > verapamil; and (4) the presence or absence of fetal calf serum had no effect on the bioavailability of chemosensitizer agents, with the exception of serum-free experiments, which showed a significant decrease in P-gp activity under the presence of PSC-833 (P = 0.05). Hence, we recommend this experimental strategy for clinical practice better to study the cellular drug resistance phenotype.

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Department of Cryobiology and Cell Therapy, Hospital Duran i Reynals, L'Hospitalet del Llobregat, Barcelona, Spain.

The study of the nuclear DNA content by flow cytometry (FCM) has been classically accomplished by selecting the nuclear population on the biparametric forward scatter (FS)-DNA fluorescence or FS-DNA fluorescence peak histograms to determine ploidy and DNA index (DI). Different cellular factors such as nuclear morphological heterogeneity of the neoplastic cells, intratumoral variability, histological origin, displasia grade, necrosis, and size of the tumoral piece analyzed constitute important problems in ploidy studies and, consequently, residual or underrepresented clones with different ploidy levels can be masked by populations with a large cell number. In the present report, an alternative methodology is proposed for aneuploidy detection, since populations coinciding with DNA content may be different with respect to morphological criteria. The discrimination of aggregates and background noise by using peak or logarithmic fluorescence signal, and backgating in side scatter (SS)/FS histograms, permits the establishment of specific bounds through complete scatterplot mapping and to distinguish between scarce or minor populations in association with small or abnormal DNA peaks. Moreover, variations in the DNA modal channel value and the peak coefficient of variation value remained unmodified, also maintaining the quality of cytometric measurement data.