Induction of telomerase activity during development of human mast cells from peripheral blood CD34+ cells: comparisons with tumor mast-cell lines.
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To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication. (+info)
High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361).
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Multiparameter flow cytometry (MFC) has the potential to allow for sensitive and specific monitoring of residual disease (RD) in acute myeloid leukemia (AML). The use of MFC for RD monitoring assumes that AML cells identified by their immunophenotype at diagnosis can be detected during remission and at relapse. AML cells from 136 patients were immunophenotyped by MFC at diagnosis and at first relapse using 9 panels of 3 monoclonal antibodies. Immunophenotype changes occurred in 124 patients (91%); they consisted of gains or losses of discrete leukemia cell populations resolved by MFC (42 patients) and gains or losses of antigens on leukemia cell populations present at both time points (108 patients). Antigen expression defining unusual phenotypes changed frequently: CD13, CD33, and CD34, absent at diagnosis in 3, 33, and 47 cases, respectively, were gained at relapse in 2 (67%), 15 (45%), and 17 (36%); CD56, CD19, and CD14, present at diagnosis in 5, 16, and 20 cases, were lost at relapse in 2 (40%), 6 (38%), and 8 (40%). Leukemia cell gates created in pretreatment samples using each 3-antibody panel allowed identification of relapse AML cells in only 68% to 91% of cases, but use of 8 3-antibody panels, which included antibodies to a total of 16 antigens, allowed identification of relapse AML cells in all cases. Thus, the immunophenotype of AML cells is markedly unstable; nevertheless, despite this instability, MFC has the potential to identify RD in AML if multiple antibody panels are used at all time points. (Blood. 2001;97:3574-3580) (+info)
Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids.
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The human parvovirus adeno-associated virus type 2 (AAV-2) possesses many features that make it an attractive vector for gene delivery in vivo. However, its broad host range may limit its usefulness and effectivity in several gene therapy applications in which transgene expression needs to be limited to a specific organ or cell type. In this study, we explored the possibility of directing recombinant AAV-2 transduction by incorporating targeting peptides previously isolated by in vivo phage display. Two putative loops within the AAV-2 capsid were examined as sites for incorporation of peptides. We tested the effects of deleting these loops and different strategies for the incorporation of several targeting peptides. The tumor-targeting sequence NGRAHA and a Myc epitope control were incorporated either as insertions or as replacements of the original capsid sequence. Viruses were assessed for packaging, accessibility of incorporated peptides, heparin binding, and transduction in a range of cell lines. Whereas recombinant viruses containing mutant capsid proteins were produced efficiently, transduction of several cell lines was significantly impaired for most modifications. However, certain mutants containing the peptide motif NGR, which binds CD13 (a receptor expressed in angiogenic vasculature and in many tumor cell lines), displayed an altered tropism toward cells expressing this receptor. Based on this work and previous studies, possible strategies for achieving in vivo targeting of recombinant AAV-2 are discussed. (+info)
Phenotypic characterization of five dendritic cell subsets in human tonsils.
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Heterogeneous expression of several antigens on the three currently defined tonsil dendritic cell (DC) subsets encouraged us to re-examine tonsil DCs using a new method that minimized DC differentiation and activation during their preparation. Three-color flow cytometry and dual-color immunohistology was used in conjunction with an extensive panel of antibodies to relevant DC-related antigens to analyze lin(-) HLA-DR(+) tonsil DCs. Here we identify, quantify, and locate five tonsil DC subsets based on their relative expression of the HLA-DR, CD11c, CD13, and CD123 antigens. In situ localization identified four of these DC subsets as distinct interdigitating DC populations. These included three new interdigitating DC subsets defined as HLA-DR(hi) CD11c(+) DCs, HLA-DR(mod) CD11c(+) CD13(+) DCs, and HLA-DR(mod) CD11c(-) CD123(-) DCs, as well as the plasmacytoid DCs (HLA-DR(mod) CD11c(-) CD123(+)). These subsets differed in their expression of DC-associated differentiation/activation antigens and co-stimulator molecules including CD83, CMRF-44, CMRF-56, 2-7, CD86, and 4-1BB ligand. The fifth HLA-DR(mod) CD11c(+) DC subset was identified as germinal center DCs, but contrary to previous reports they are redefined as lacking the CD13 antigen. The definition and extensive phenotypic analysis of these five DC subsets in human tonsil extends our understanding of the complexity of DC biology. (+info)
Transient myeloproliferative disorder and acute myeloid leukemia in Down syndrome. An immunophenotypic analysis.
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Immunophenotypic analysis of transient myeloproliferative disorder (TMD) and acute myeloid leukemia (AML) using multiparameter flow cytometry might provide insight into their relationship. We retrospectively analyzed the expression of multiple lymphoid, myelomonocytic, and megakaryocytic antigens on blast proliferations in 18 patients with Down syndrome (DS; AML, 9; TMD, 9). The AMLs and TMDs shared several immunophenotypic characteristics. Blasts in all expressed CD45, CD38, and CD33; most AMLs and all TMDs were CD36+; and the majority expressed CD41 and CD61, suggesting megakaryocytic differentiation. The majority of cases were CD34+, CD14-, and CD64-. There was aberrant expression of the T-cell-associated antigen CD7 in most AMLs and TMDs. CD56 was expressed aberrantly in 5 AMLs and 7 TMDs. The major difference between the disorders was the pattern of expression of myeloid markers CD11b and CD13; each was expressed in 8 AMLs but only 2 TMDs. Blasts were HLA-DR-positive in 3 AMLs vs 7 TMDs. Blasts in TMD and AML in DS have a characteristic immunophenotype distinct from AML in other settings. The immunophenotypic similarities suggest a biologic relationship between the disorders; however, distinct immunophenotypic differences also were observed. (+info)
Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis.
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Two renal cell carcinoma cell lines (49RC 43STR and 75RC 2STR) were characterized by detection of the cell surface proteins: CD44(var), intercellular adhesion molecule-1 (ICAM-1), urokinase-type plasminogen activator (uPA) and its receptor and aminopeptidase N (APN). To detect their localization the immunoluminescent technique was used. In addition, the enzyme activity of uPA and APN was investigated in cell suspensions as well as in monolayers. The latter procedure was more advantageous since the additional use of HPLC permits a single registration of the fluorescent hydrolysis-product AMC (7-amino-4-methylcoumarin) without interference by cellular autofluorescence or non-reacted fluorescent substrate. Unlike 75RC 2STR, the cell line 49RC 43STR expressed high levels of uPA and APN. Contrary to that the cell line 75RC 2STR expressed high levels of ICAM-1 and CD44(v6), whereas 49RC 43STR showed a low level of ICAM-1 and no distinct light signal with anti-CD44(v6). The uPA activity was measured directly as well as indirectly (via plasmin) with the substrate Z-Gly-Gly-Arg-AMC. Both activator and plasmin activity were inhibited by D-Val-Phe-Lys-CMK and phenylmethylsulfonyl fluoride. The anti-catalytic antibody to uPA and that to uPA receptor were found to be inhibiting the uPA activity in a concentration-dependent manner. APN activity was assayed using alanine-p-nitroanilide. Peptidase activity was effectively inhibited by 1,10-phenanthroline and partly inhibited by ethylenediamine-tetraacetic acid. (+info)
Molecular determinants of species specificity in the coronavirus receptor aminopeptidase N (CD13): influence of N-linked glycosylation.
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Aminopeptidase N (APN), a 150-kDa metalloprotease also called CD13, serves as a receptor for serologically related coronaviruses of humans (human coronavirus 229E [HCoV-229E]), pigs, and cats. These virus-receptor interactions can be highly species specific; for example, the human coronavirus can use human APN (hAPN) but not porcine APN (pAPN) as its cellular receptor, and porcine coronaviruses can use pAPN but not hAPN. Substitution of pAPN amino acids 283 to 290 into hAPN for the corresponding amino acids 288 to 295 introduced an N-glycosylation sequon at amino acids 291 to 293 that blocked HCoV-229E receptor activity of hAPN. Substitution of two amino acids that inserted an N-glycosylation site at amino acid 291 also resulted in a mutant hAPN that lacked receptor activity because it failed to bind HCoV-229E. Single amino acid revertants that removed this sequon at amino acids 291 to 293 but had one or five pAPN amino acid substitution(s) in this region all regained HCoV-229E binding and receptor activities. To determine if other N-linked glycosylation differences between hAPN, feline APN (fAPN), and pAPN account for receptor specificity of pig and cat coronaviruses, a mutant hAPN protein that, like fAPN and pAPN, lacked a glycosylation sequon at 818 to 820 was studied. This sequon is within the region that determines receptor activity for porcine and feline coronaviruses. Mutant hAPN lacking the sequon at amino acids 818 to 820 maintained HCoV-229E receptor activity but did not gain receptor activity for porcine or feline coronaviruses. Thus, certain differences in glycosylation between coronavirus receptors from different species are critical determinants in the species specificity of infection. (+info)
Erythropoietin-dependent transformation of myelodysplastic syndrome to acute monoblastic leukemia.
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Acute monoblastic leukemia (acute myeloid leukemia [AML], French-American-British type M5a) with leukemia cutis developed in a patient 6 weeks after the initiation of erythropoietin (EPO) therapy for refractory anemia with ringed sideroblasts. AML disappeared from both marrow and skin after the discontinuation of EPO. Multiparameter flow cytometric analysis of bone marrow cells demonstrated coexpression of the EPO receptor with CD45 and CD13 on the surface of blasts. The incubation of marrow cells with EPO, compared to without, resulted in 1.3- and 1.6-fold increases, respectively, in tritiated thymidine incorporation and bromodeoxyuridine incorporation into CD13(+) cells. Clinical and laboratory findings were consistent with the EPO-dependent transformation of myelodysplastic syndrome (MDS) to AML. It is concluded that leukemic transformation in patients with MDS treated with EPO may be EPO-dependent and that management should consist of the discontinuation of EPO followed by observation, if clinically feasible. (+info)