G-CSF increases the number of peripheral blood dendritic cells CD16+ and modifies the expression of the costimulatory molecule CD86+.

Dendritic cells (DC) play a key role in initiating immune reactions after allogeneic stem cell transplantation. The two main peripheral blood DC populations are myeloid (DC1) and lymphoplasmacytoid (DC2). A new subset of myeloid DC, expressing CD16, has been identified. We analyzed the number and CD86 expression of DC subsets in peripheral blood of 18 healthy donors, before and after granulocyte colony-stimulating factor (G-CSF) and in the inoculum of allogeneic peripheral blood transplants (allo-PBT; n=100) and allogeneic bone marrow transplants (allo-BMT; n=22). Granulocyte colony-stimulating factor administration increased the median number of DC1 (P=0.0007), of DC2 (P<0.0001) and of DC CD16+ (P=0.0001). Granulocyte colony-stimulating factor administration was also associated with a significant decrease of CD86 expression on DC1 (P=0.0003) and with a trend for an increase on DC CD16+ (P=0.07). Recipients of allo-PBT received similar quantities of DC1 and higher doses of DC2 and DC CD16+ than recipients of allo-BMT (P=0.5; P=0.0001; P<0.0001, respectively). Granulocyte colony-stimulating factor modifies the number of DC in peripheral blood and the expression of the costimulatory molecule CD86. This resulted in a different composition of DC2 and especially of DC CD16+ in the harvests, which might explain some of the differences observed in allogeneic reactions after allo-PBT with respect to allo-BMT.

Redefining the significance of aneuploidy in the prognostic assessment of colorectal cancer.

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.

Preparation of immunoliposomes bearing poly(ethylene glycol)-coupled monoclonal antibody linked via a cleavable disulfide bond for ex vivo applications.

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.

Retroviral gene transfer into human hematopoietic cells: an in vitro kinetic study.

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.

Preparation of immunoliposomes directed against CD34 antigen as target.

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.

Comparative effect of verapamil, cyclosporin A and SDZ PSC 833 on rhodamine 123 transport and cell cycle in vinblastine-resistant Chinese hamster ovary cells overexpressing P-glycoprotein.

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.

Flow cytometric analysis of P-glycoprotein function using rhodamine 123.

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.

Is rhodamine 123 an appropriate fluorescent probe to assess P-glycoprotein mediated multidrug resistance in vinblastine-resistant CHO cells?

Cellular drug resistance, which involves several mechanisms such as P-glycoprotein (P-gp) overexpression, kinetic and metabolic quiescence, or the increase in the intracellular levels of glutathione, limits the effectiveness of cancer treatment. It has been reported that functional assessment of the cationic dye rhodamin 123 (Rho123) efflux reveals accurately the drug-resistant phenotype. To study cellular drug resistance, we have obtained a CHO-K1 derived cell line resistant to vinblastine by means of multistep selection. This cell line (CHOVBR) displays high reactivity with a monoclonal antibody (MAb) (C219) directed against an internal domain of P-gp, and an active Rho123 efflux, as shown by parallel flow cytometric and fluorometric assays. However, under similar experimental conditions, the drug-sensitive parental cell line CHO-K1 (as well as the myeloblastic KG1 and KG1a cell lines), was also able to pump Rho123 out. These parental CHO-K1 cells had a very low reactivity against the C219 Mab, as confirmed by Western blot analysis. Both vinblastine and verapamil inhibited Rho123 efflux in CHO-K1 cells, but had no effect on CHOVBR cultures. Also, deprivation of vinblastine for one month did not affect Rho123 efflux in these cells. Our results suggest that the activity of P-gp appears to be essential, but not sufficient to confer drug resistance, and that Rho123-based functional assays of drug resistance should be evaluated for each cellular experimental model.

Relevance of forward scatter and side scatter in aneuploidy detection by flow cytometry.

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.

Flow cytometry analysis of pituitary adenomas.

UNLABELLED: Using flow cytometry, DNA content and index, and/or proliferative capacity (measuring proliferating cell nuclear antigen PCNA) in operated pituitary tumors, control pituitaries obtained at necropsy, and experimental pituitary hyperplasia induced in rats were analyzed. Simultaneous measurement of cell ploidy and proliferation differentiated normal pituitary (diploid DNA index and negative PCNA) from pituitary hyperplasia (diploid DNA index with intensely positive PCNA, between 30 and 72% of cells). In the tumors 83% (19/ 23) were positive for PCNA (between 3 and 84%) and 73% (17/23) aneuploid; only 1 tumor was diploid and negative for PCNA. CONCLUSIONS: Differentiation between normal and abnormal (neoplastic or hyperplastic) pituitary is possible by flow cytometry, but in the adenomas no correlation with postoperative clinical outcome was observed.

CD34+ cell positive selection from mobilized peripheral blood by an indirect immunomagnetic method: effect of the type of mobilization and assessment of tumor depletion ability.

Mobilized peripheral blood has been shown to be a suitable source of hematopoietic progenitor cells for autologous transplantation in oncologic patients. However, tumor cell contamination can potentially occur, although to a lesser extent than in the bone marrow. CD34+ cell positive selection has been developed as a system for the ex vivo purging of remaining tumor cells when used in mobilized peripheral blood. This method has shown a lower purification potential than that obtained with bone marrow or cord blood. The reason for this is not clear, but different groups have tried to improve the purity and yield of the positive selection on mobilized peripheral blood by predepletion of nonlymphoid cell populations, since they can induce nonspecific binding. The present study was designed to test an indirect immunomagnetic CD34+ cell selection method to make it reproducible, feasible, and effective for purging mobilized peripheral blood. Twenty-nine samples from mobilized peripheral blood were tested. The median starting CD34+ percentage was 0.8 (0.3%-4.2%), and the median final purity was 87% (32.7%-99.7%), with a median yield of 44.8% (15%-83.5%). The highest purity was reproducibly achieved when the starting percentage of CD34+ cells was higher than 0.65% (median purity 93.7, range 81%-99.7%, CV 6%) on samples obtained from patients primed with chemotherapy alone or chemotherapy plus recombinant human granulocyte-colony stimulating factor. No relation was found between the content of contaminating nucleated cells and the final CD34+ cell purity. This method showed a depletion capacity, assessed by PCR on samples contaminated with K562 leukemic cells, of about 3 logs. These results indicate that this indirect immunomagnetic method can produce high purity CD34+ cell fractions from mobilized peripheral blood with a high efficiency of tumor depletion.