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PubMed2005

A new class of 5-fluoro-2'-deoxyuridine prodrugs conjugated with a tumor-homing cyclic peptide CNGRC by ester linkers: synthesis, reactivity, and tumor-cell-selective cytotoxicity.

PURPOSE: Tumor-targeting prodrugs of 5-fluoro-2'-deoxyuridine (5-FdUrd), which are chemical conjugations of 5-FdUrd with a tumor-homing cyclic peptide CNGRC by succinate and glutarate linkers, were synthesized to investigate the structural effects of linkers on the hydrolytic release of 5-FdUrd and the tumor-cell-selective cytotoxicity. METHODS: A solid phase synthesis method was used to produce 5-FdUrd prodrugs. The kinetics and efficiency of hydrolytic 5-FdUrd release from the prodrugs were investigated in phosphate buffer (PB), fetal bovine serum (FBS), HT-1080 cell lysate, MDA-MB-231 cell lysate, and MEM containing 10% FBS. The tumor-cell-selective cytotoxicity of prodrugs was evaluated by an MTT method. RESULTS: Two tumor-targeting prodrugs CNF1 and CNF2 bearing 5-FdUrd conjugated with a common cyclic peptide CNGRC by succinate and glutarate linkers, respectively, and their control compounds CN1 and CN2 without 5-FdUrd moiety were synthesized and identified. CNF1 underwent hydrolysis to release 5-FdUrd more rapidly and efficiently than CNF2. Both prodrugs were of lower cytotoxicity compared to 5-FdUrd, showing more selective cytotoxicity toward APN/CD13 positive cells (HT-1080) than toward APN/CD13 negative cells (HT-29, MDA-MB-231). CONCLUSIONS: A new class of tumor-targeting 5-FdUrd prodrugs CNF1 and CNF2 were successfully synthesized. These prodrugs targeted a tumor marker APN/CD13 to cause tumor-cell-selective cyctotoxicity due to 5-FdUrd release, the rate of which could be controlled by the structure of ester linker.

Antineoplastic Agents↗

PubMed1986

Verapamil-mediated sensitization of doxorubicin-selected pleiotropic resistance in human sarcoma cells: selectivity for drugs which produce DNA scission.

The effects of verapamil on the cytotoxicity and accumulation of multiple drugs were studied in a model of pleiotropic resistance generated by doxorubicin (DOX) selection of the human sarcoma cell line MES-SA. The in vitro sensitivity of the DOX-resistant variant (named Dx5), which is 50- to 100-fold resistant to DOX compared to MES-SA, was enhanced approximately 7-fold by verapamil (3 micrograms/ml). In addition, the cytotoxicity of several agents to which the Dx5 line displays cross-resistance, i.e., daunorubicin, dactinomycin, mitoxantrone, and etoposide, was also enhanced 2- to 14-fold by verapamil. These agents share the properties of DNA intercalation and/or interaction with topoisomerase II. In contrast, verapamil did not alter the sensitivity of Dx5 to several other agents to which cross-resistance had been demonstrated, i.e., vincristine, vinblastine, colchicine, mitomycin C, and melphalan; nor did verapamil enhance the cytotoxicity of DOX or other agents against the DOX-sensitive parent, MES-SA. The sensitizing effect of verapamil did not correlate well with its effects on intracellular drug accumulation. [14C]DPX accumulation was increased by 30-40% in Dx5 but not in MES-SA cells in the presence of verapamil. [3H]Vinblastine accumulation was increased by 24-72% in both MES-SA and Dx5 cells in the presence of verapamil, although cytotoxicity of the Vinca alkaloids was not affected. In this human sarcoma model of DOX-selected pleiotropic resistance, verapamil partially reversed the resistance to DOX, as well as four of the nine drugs for which cross-resistance had been demonstrated in Dx5. The potentiation by verapamil of the cytotoxicity of some but not all of these antitumor agents suggests that factors other than altered drug transport may be responsible. The pattern of sensitization, restricted to agents which produce DNA strand scission by interaction with topoisomerase II, suggests that verapamil may be acting to promote the formation or inhibit the repair of such DNA strand breaks.

Animals↗

PubMed2000

Depletion of alloreactive donor T cells using immunomagnetic cell selection.

Donor T cells support both engraftment and immune reconstitution after allogeneic BMT. Moreover, they may exert potent anti-tumor effects (graft-versus-leukemia, GVL), which are used for adoptive immunotherapy. On the other hand, infusion of allogeneic T cells is frequently associated with the manifestation of immune reactions against healthy tissue, which may lead to life-threatening graft-versus-host disease (GVHD). To overcome this problem, we developed a new strategy for the exclusive depletion of alloreactive cells from donor leukocytes. We activated donor T cells by co-cultivation with a stroma layer of recipient cells and analyzed activation kinetics of CD3+, CD4+ and CD8+ T cells. Based on these data, activated cells were then depleted based on expression of activation-induced antigens using magnetic cell sorting (MACS). Alloreactivity of donor T cells was remarkably decreased after depletion of cells expressing either CD25 or CD69, as was shown in suitable in vitro assays. The lowest level of alloreactivity was found when both CD25- and CD69-positive cells were depleted. Importantly, depleted cell fractions preserved reactivity against third-party cells. In summary, we found that MACS-based ex vivo depletion of alloreactive cells may be a suitable way to prevent GVHD and therefore improve allogeneic BMT and adoptive immunotherapy.

Antigens, CD↗

PubMed1992

Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool.

The B cells of immunized animals can be used as a source of variable region (V) gene libraries. Such libraries offer a way of making antibodies directly in bacteria: rearranged V genes are amplified using the polymerase chain reaction, cloned and expressed as soluble fragments in bacteria, and then screened for antigen binding. Here we have used a model system to investigate antigen-selected B cells as a source of V gene libraries. Mice were immunized with (4-hydroxyl-3-nitrophenyl)acetyl (NP)-chicken gammaglobulin, and the splenocytes harvested seven days after primary immunization. We prepared a heavy chain variable (VH) gene library from the DNA of cells selected on antigen-coated magnetic beads, and two other libraries from the DNA or mRNA of unselected cells. The VH gene libraries were combined with the V lambda 1 gene (as this light chain dominates the primary response to NP), expressed as Fv fragments in Escherichia coli and screened for binding to (4-hydroxy-3-iodo-5-nitrophenyl)acetyl-bovine serum albumin. The frequency of antigen-binding clones was much greater (greater than 50 fold) in the library from the DNA of antigen-selected cells (17/282) or from the mRNA of unselected cells (29/282) compared to the DNA from unselected cells (0/940). Sequencing of the antigen-binding clones revealed that they almost invariably used the V-186.2 heavy chain, as expected from analysis of primary response hybridomas. The D segments from the mRNA library were entirely DFL16.1 (29/29), as in primary response hybridomas, whereas those from the DNA of selected cells were more diverse, using in addition to DFL16.1, other D segments (5/17) as in later response hybridomas. This suggests that the DNA library from selected cells is derived at least in part from cells destined for the memory compartment. Given the long life of memory cells, they may prove a useful source of antibody libraries in the absence of recent immunization.

Amino Acid Sequence↗

PubMed2000

Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols.

BACKGROUND: Cord blood is a useful source of HPCs for allogeneic transplantation. HPC ex vivo expansion of a cord blood graft has been proposed as a way to increase the speed of engraftment and thus to reduce the occurrence of transplantation-related complications. OBJECTIVE: The purpose of this study was to optimize a method for CD34+ cell selection of thawed cord blood grafts under clinical grade conditions, intended for application in a static, serum-free expansion culture. MATERIAL AND METHODS: Twelve samples were thawed and washed with dextran, albumin, and rHu-deoxyribo-nuclease I (RHu-DNase) to avoid clumping. CD34+ cells were selected by using a sensitized immunomagnetic bead and 9C5 MoAb complex. A buffer containing rHu-DNase, citrate, albumin, and immunoglobulin in PBS was used during the procedure. CD34+ cells were eluted and detached by using an immunomagnetic cell selection device. Cells from the enriched fraction were cultured for 6 days in serum-free medium supplemented with rHu-SCF, rHu-IL-3, rHu fetal liver tyrosine kinase 3 ligand, and rHu thrombopoietin (50 ng/mL each). Cells were expanded in well plates and in two semipermeable bags. RESULTS: A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yielding a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ cells was 32 +/- 10 percent. CFU-GM and BFU-E/CFU-Mixed recoveries were 33 +/- 15 percent and 27 +/- 12 percent, respectively. CD34+ cells obtained were functionally comparable with fresh CD34+ cells selected for clonogenic potential. The capacity for expansion was not significantly different in the two types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD34+ cells and 42 +/- 19-fold for overall colonies. The expansion rates observed in wells were significantly superior to those obtained in bags. CONCLUSION: The feasibility of a clinical-scale cord blood selection procedure based on a direct immunomagnetic method after thawing, followed by an ex vivo expansion culture using semipermeable bags, is shown. After 6 days of expansion, it was possible to generate a 9-fold increase in CD34+ cells, a 6-fold increase in CFU-GM and a 13-fold increase in BFU-E/CFU-Mixed colonies.

Antigens, CD34↗

PubMed2002

Responses of spectrally selective cells in macaque area V2 to wavelengths and colors.

We have recorded from wavelength-selective cells in macaque monkey visual area V2, interposed between areas V1 and V4 of the color-specialized pathway, to learn whether their responses correlate with perceived colors or are determined by the wavelength composition of light reflected from their receptive fields. All the cells we recorded from were unselective for the orientation and direction of motion of the stimulus, and all were histologically identified to be in the thin cytochrome oxidase stripes. Using multi-colored "Mondrian" scenes of the appropriate spatial configuration, areas of different color were placed in the receptive field of each cell and the entire scene illuminated by three projectors, passing long-, middle-, and short-wave light, respectively, in various combinations. Our results show that wavelength-selective cells in V2 respond to an area of any color depending on whether or not it reflects a sufficient amount of light of their preferred wavelength. In addition, the responses of a third of the cells tested were also influenced by the wavelength composition of their immediate surrounds, thus signaling the result of a local spatial comparison with respect to the amount of their preferred wavelength present. The responses of all also depended on the sequence with which their receptive fields were illuminated with light of the three different wavebands: cells were activated when there was an increase (and inhibited when there was a decrease) in the amount of their preferred wavelength with respect to the other two; the temporal route taken was therefore a determining factor, and, depending on it, cells would either respond or not to a particular combination of wavelengths. We conclude that although spatiotemporal wavelength comparisons are taking place in the color-specialized subdivisions of area V2, the determination of complete color-constant behavior at the neuronal level requires further processing, in other areas.

Animals↗

PubMed1991

The enzyme-inhibitor approach to cell-selective labelling. III. Sulphonamide inhibitors of carbonic anhydrase as carriers for red cell labelling.

Selective radiolabelling of red blood cells via an enzyme-inhibitor approach represents a novel method in diagnostic nuclear medicine. Current problems in blood pool labelling could be overcome by using selective sulphonamide inhibitors as carriers. Red cell carbonic anhydrase is identified as an ideal target enzyme for such an approach. A brief review of the target enzyme is presented together with the screening of a series of synthesised sulphonamide inhibitors. p-Iodobenzenesulphonamide, 4-[(4-iodophenyl)thio]benzenesulphonamide and 5-(4-bromophenyl)sulphonyl]thiophene-2-sulphonamide were found to be particularly potent, reversible, lipophilic inhibitors of carbonic anhydrase, characteristics that warrant their further investigation as potential carriers. 4-Iodo-3-(iodoacetamido)benzenesulphonamide was a moderate inhibitor but caused relatively fast irreversible inactivation, making it a candidate for longer term studies.

Animals↗

PubMed1977

Renal enzymes in kidney cells selected by D-valine medium.

The ability of D-valine medium to inhibit fibroblasts in cultures derived from kidneys of various mammals has enabled the selective proliferation of epithlial cells in the absence of fibroblast overgrowth. Studies of these selected epithelial cells have demonstrated the presence of D-amino acid oxidase, carbonic anhydrase, high levels of alkaline phosphatase and the renal specific pattern of lactate dehydrogenase. The presence of these renal enzymes suggests that the selected epithelial cells are of renal tubular origin and indicates that these differentiated functions are retained in cultured cells.

Alkaline Phosphatase↗

PubMed2001

Severe infections after allogeneic peripheral blood stem cell transplantation: a matched-pair comparison of unmanipulated and CD34+ cell-selected transplantation.

BACKGROUND AND OBJECTIVES: T-cell depletion of the graft delays immune recovery following allogeneic peripheral blood stem cell transplantation (PBSCT), but it is not clear whether it actually increases the risk of severe infections after the transplant. DESIGN AND METHODS: We have compared the occurrence of severe infections following 162 CD34+ cell-selected allogeneic PBSCT and 162 unmanipulated PBSCT (CD34+ and UM groups, respectively) from HLA-identical siblings. RESULTS: The probability of infection-related mortality (IRM) was 22% in the UM group and 31% in the CD34+ group (log-rank, p=0.2). In multivariate analyses only the use of fluconazole prophylaxis showed a protective effect on IRM in the whole set of patients, while in both transplant groups the most significant factor was the development of moderate-to-severe graft-versus-host disease (GVHD). The probability of developing cytomegalovirus (CMV) infection was 42% in the UM group and 59% in the CD34+ group (p=0.002), with no differences in CMV disease (10% and 9%, respectively). Multivariate analysis of CMV infection identified three variables associated with a higher risk in the whole set of patients: CMV positive serostatus, CD34+ transplant group and recipient age above 40 years. The development of moderate-to-severe GVHD was a significant factor only in the UM group. Disseminated varicella-zoster virus infection was more common in the CD34+ group (19% and 12%, p=0.05), as were early (< 30 days post-transplant) severe bacterial infections (28% vs 14%, p=0.002). Invasive fungal infections and pneumonias of unknown origin did not differ between groups. INTERPRETATION AND CONCLUSIONS: Our results do not show a significant increase in the risk of dying from an opportunistic infection with CD34+-PBSCT, but the risk of CMV infection is increased, with no differences in CMV disease or mortality attributable to CMV. There is an additive effect on IRM of developing moderate-to-severe GVHD (acute or chronic) following CD34+-PBSCT, and in this subset of patients maximum efforts for the prevention and early treatment of opportunistic infections should be pursued.

Adolescent↗

PubMed1999

Mutant cells selected during persistent reovirus infection do not express mature cathepsin L and do not support reovirus disassembly.

Persistent reovirus infections of murine L929 cells select cellular mutations that inhibit viral disassembly within the endocytic pathway. Mutant cells support reovirus growth when infection is initiated with infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly formed following proteolysis of viral outer-capsid proteins. However, mutant cells do not support growth of virions, indicating that these cells have a defect in virion-to-ISVP processing. To better understand mechanisms by which viruses use the endocytic pathway to enter cells, we defined steps in reovirus replication blocked in mutant cells selected during persistent infection. Subcellular localization of reovirus after adsorption to parental and mutant cells was assessed using confocal microscopy and virions conjugated to a fluorescent probe. Parental and mutant cells did not differ in the capacity to internalize virions or distribute them to perinuclear compartments. Using pH-sensitive probes, the intravesicular pH was determined and found to be equivalent in parental and mutant cells. In both cell types, virions localized to acidified intracellular organelles. The capacity of parental and mutant cells to support proteolysis of reovirus virions was assessed by monitoring the appearance of disassembly intermediates following adsorption of radiolabeled viral particles. Within 2 h after adsorption to parental cells, proteolysis of viral outer-capsid proteins was observed, consistent with formation of ISVPs. However, in mutant cells, no proteolysis of viral proteins was detected up to 8 h postadsorption. Since treatment of cells with E64, an inhibitor of cysteine-containing proteases, blocks reovirus disassembly, we used immunoblot analysis to assess the expression of cathepsin L, a lysosomal cysteine protease. In contrast to parental cells, mutant cells did not express the mature, proteolytically active form of the enzyme. The defect in cathepsin L maturation was not associated with mutations in procathepsin L mRNA, was not complemented by procathepsin L overexpression, and did not affect the maturation of cathepsin B, another lysosomal cysteine protease. These findings indicate that persistent reovirus infections select cellular mutations that affect the maturation of cathepsin L and suggest that alterations in the expression of lysosomal proteases can modulate viral cytopathicity.

Animals↗

PubMed1998

T cell selection during the evolution of CD8+ T cell memory in vivo.

Memory T cell responses are frequently highly restricted in terms of receptor usage. How and when such clonotypic dominance is established remains poorly understood. Here we have investigated the evolution of the T cell responses to an epitope from Epstein-Barr virus (EBV), (FLRGRAYGL), by analyzing TCR use of clones specific for this epitope, derived from peripheral blood mononuclear cells taken from individuals early during primary EBV infection and up to 3 years later. We show that, in a given individual, particular T cell clonotypes are selected early during the primary response to this epitope and that the same clonotypes dominate the late memory response. In one individual direct analysis of HLA-B8-restricted FLRGRAYGL-specific T cells, isolated from peripheral blood lymphocytes taken during primary EBV infection using a tetrameric MHC-peptide complex, confirmed the early selection of the dominant clonotypes.

Amino Acid Sequence↗

PubMed1988

Laminar organization and contrast sensitivity of direction-selective cells in the striate cortex of the Old World monkey.

The directional preference of neurons sampled from all layers of the striate cortex was determined using the responses to drifting grating stimuli of optimal spatial and temporal frequency. In addition, contrast sensitivity as a function of spatial frequency was measured and from the resulting spatial contrast sensitivity function the peak contrast sensitivity and optimal spatial frequency were obtained. The distribution of directionally selective cells showed a distinct laminar pattern. Upper layer 4 (4a, 4b, and 4c alpha) and layer 6 were the only cortical layers with neurons that showed a pronounced preference for the direction of stimulus motion. The directionally selective cells in these layers are among those with the highest contrast sensitivities but had optimal spatial frequencies that were confined to the low and middle range of the optimal spatial frequency distribution. These findings suggest that the directionally selective cells may fall into at least 2 distinct populations, which may be the first stages in the visual pathway that correspond to those channels, inferred from psychophysical experiments, that underlie the detection of movement.

Animals↗

PubMed2004

Origins of cell selectivity of cationic steroid antibiotics.

A key factor in the potential clinical utility of membrane-active antibiotics is their cell selectivity (i.e., prokaryote over eukaryote). Cationic steroid antibiotics were developed to mimic the lipid A binding character of polymyxin B and are shown to bind lipid A derivatives with affinity greater than that of polymyxin B. The outer membranes of Gram-negative bacteria are comprised primarily of lipid A, and a fluorophore-appended cationic steroid antibiotic displays very high selectivity for Gram-negative bacterial membranes over Gram-positive bacteria and eukaryotic cell membranes. This cell selectivity of cationic steroid antibiotics may be due, in part, to the affinity of these compounds for lipid A.

Anti-Bacterial Agents↗

PubMed2004

Medical devices; immunology and microbiology devices; classification of the immunomagnetic circulating cancer cell selection and enumeration system. Final rule.

The Food and Drug Administration (FDA) is classifying the Immunomagnetic Circulating Cancer Cell Selection and Enumeration System device into class II (special controls). The special control that will apply to the device is the guidance document entitled "Class II Special Controls Guidance Document: Immunomagnetic Circulating Cancer Cell Selection and Enumeration System." The agency is taking this action in response to a petition submitted under the Federal Food, Drug, and Cosmetic Act (the act) as amended by the Medical Device Amendments of 1976 (the amendments), the Safe Medical Devices Act of 1990 (the SMDA), the Food and Drug Administration Modernization Act of 1997 (FDAMA), and the Medical Device User Fee and Modernization Act of 2002 (MDUFMA). The agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is publishing a notice of availability of a guidance document that is the special control for this device.

Cell Count↗

PubMed1998

Sequential CD34+ and CD4+ cell selection from leukapheresis components.

We tested the feasibility of sequential selection of CD34+ and CD4+ cell-enriched fractions from aliquots of five autologous leukapheresis components. CD34+ cells were selected from a median 8.0 x 10(8) mononuclear cells using the CellPro CEPRATE LC cell separation system. All cells in the CD34-depleted fraction (median 4.8 x 10(8), range 0.6-10.0 x 10(8) were then incubated with the appropriate antibodies for CD4+ cell selection and passed through a second LC column. Median target cell purities of the CD34+ cell-enriched fraction and CD4+ cell-enriched fraction were 90.5% and 86.0%, respectively. This study demonstrates that high purity CD34+ and CD4+ cell-enriched fractions can be isolated sequentially from leukapheresis components. In addition, CD8+ lymphocytes, implicated in graft-versus-host disease, were depleted in the course of both positive selection procedures. This approach could decrease the number of donor procedures by providing separate CD34(+)-enriched and CD4(+)-enriched populations for allogeneic peripheral blood progenitor cell transplantation and subsequent donor lymphocyte infusion from the same leukapheresis component.

Antigens, CD34↗

PubMed1997

The relationship of a novel drug-resistant phenotype in C3H10T1/2 cells selected with alkylating agents to neoplastic transformation and ATP metabolism.

We investigated the resistance to alkylating agents in parental, drug-selected and neoplastically transformed C3H10T1/2 (10T1/2) murine fibroblasts. Similar levels of resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were observed in cells selected for resistance to MNNG or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) as well as in cells transformed by a single treatment with MNNG. Surprisingly, neither the levels of O6-alkylguanine-DNA alkyltransferase (AT) nor glutathione-S-transferase (GST) were altered in drug-resistant cells. In contrast, changes in ATP metabolism were observed in both transformed and MNNG-selected cells after treatment with MNNG. Specifically, 3 h after treatment with 5 microg/ml MNNG, ATP levels decreased by 85% and 74% in MNNG-selected and transformed cells, respectively, compared to only a 28% decrease in parental cells. Therefore, rather than contributing to cell sensitivity to alkylating agents, the ability to rapidly utilize ATP and tolerate resulting decreases in ATP levels may in some cases play a role in protection from the cytotoxic effects of alkylating agents.

Adenosine Triphosphate↗

PubMed2003

Cathepsin B Is Inhibited in Mutant Cells Selected during Persistent Reovirus Infection.

Persistent reovirus infections of murine L929 (L) fibroblast cells select mutant (LX) cells that do not support proteolytic disassembly of reovirus virions within the endocytic pathway. To better understand the function and regulation of endocytic proteases, we conducted experiments to define the block to reovirus disassembly displayed by LX cells. In contrast to parental L cells, mutant LX cells harbor defects that interfere with the maturation and activity of cathepsin B and cathepsin L but not cathepsin H. The cDNAs encoding cathepsin B and cathepsin L in L cells are identical to those in LX cells, indicating that LX cells manifest an extrinsic block to the function of these enzymes. Mixed lysates of L cells and LX cells lack activity of both cathepsin B and cathepsin L, suggesting the presence of an inhibitor of cathepsin function in LX cells. A cathepsin B-green fluorescent protein (GFP) fusion protein expressed in L cells and purified by immunoprecipitation retains cathepsin B activity, whereas cathepsin B-GFP expressed in LX cells does not. However, activity of cathepsin B-GFP expressed in LX cells can be recovered by incubating the immunoprecipitate with L cell lysate followed by immunoprecipitation, providing further evidence that LX cells express a cathepsin inhibitor. Native-gel electrophoresis and gel filtration chromatography demonstrate that, in both cell lines, the double-chain form of cathepsin B is sequestered in a large molecular weight complex that renders this form of the enzyme inactive. Alteration of this sequestration complex appears to be responsible for inhibition of cathepsin B in LX cells. These findings suggest that cathepsins can be regulated within the endocytic pathway. Moreover, this regulation influences host cell susceptibility to intracellular pathogens.

Animals↗

PubMed2004

Impact of leukapheresis cell composition on immunomagnetic cell selection with the Baxter Isolex 300i device: a statistical analysis.

Immunomagnetic cell selection (ICS) of CD34(+) cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34(+) ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34(+) cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34(+) cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34(+) cells (>500 x 10(6)) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

Adolescent↗