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Caratterizzazione degli epitopi del CD34 ed analisi dei recettori per fattori di crescita nelle cellule staminali emopoietiche
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Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic MPO and lysozyme in normal and leukemic cells
No full textUsing a direct one-color (fluorescein isothiocyanate; FITC) staining method with a Facscan flow cytometer, we evaluated the intracellular expression of two granular constituents of myeloid cells [myeloperoxidase (MPO) and lysozyme] on leukemic cells from 21 patients with acute myeloid leukemia (AML), and 6 patients with acute lymphoblastic leukemia (ALL). Three different permeabilization techniques were used [FACS Lysing Solution (FLy), B.Dis; Ortho-PermeaFix (OPF); Fix and Perm (F&P), Caltag] prior to monoclonal antibody (McAb) staining, in order to verify the specificity and the sensitivity of the three labelling methods towards the two model antigens. Peripheral blood cells from 15 healthy subjects and Ortho Absolute Control served as controls. Data were expressed as percentage of positivity, net fluorescence intensity, ratio between mean fluorescence intensity (MFI) of positive cells and that of isotypic controls (P/N ratio; evaluated in both geometric and arithmetic scale), and, in 12 representatives cases (7 AML, 5 normal samples), in the form of both molecules of equivalent soluble fluorochromes (MESF) and antibody binding capacities (ABC). As far as the antigenic expression of MPO and lysozyme in normal samples is concerned, F&P resulted, in our hands, in the most specific and sensitive staining, followed by FLy solution and OPF, which showed positivity for MPO, and, to lesser extent, for lysozyme in a considerable manner of lymphocytes (means 64% and 54%, respectively, for OPF and FLy; range of ABC/cell: 0.9-5.2 x 10(3)) obtained from healthy subjects. With the reference F&P permeabilizing solution, 90% and 80% of FAB M1-M5 cases were found to be positive for MPO and lysozyme, respectively. However, M1, M2, and M3 AML FAB (French-American-British) subvarieties were characterized by a brighter expression for MPO (mean ABC/cell: 89 x 10(3)) than that of lysozyme (mean ABC/cell: 12.5 x 10(3D)), whereas blast cells from patients with M5a FAB subtypes showed higher levels of lysozyme (mean ABC/cell: 65 x 10(3)) than that of MPO (mean ABC/cell: 0.1 x 10(3)). One of five cases of FAB MO AML showed a dull positivity for MPO-7 McAb. Patients with ALL were MPO and lysozyme negative using both F&P and FLy reagents, although a certain degree of positivity was documented in some cases with OPF. Taking these data together, it can be stated that the use of anti-MPO McAbs may be of great value for the diagnosis and monitoring of acute leukemia and, along with lysozyme McAb, can provide useful information in the distinction of myeloid from monocytic leukemias and in the lineage assignment of apparently biphenotypic forms. However, the methodology used for the detection of these myeloid-associated antigens is critical for a correct interpretation of cytofluorimetric data and should be taken into account when evaluating data coming from multicenter trials dealing with leukemias. A standardization of cytofluorimetric analysis of intracellular antigens is needed in order to improve the reproducibility and comparability of results in multicenter studies
Immunophenotype definition of the stem/ progenitor cell compartment. Implications for the identification of blast cells in acute leukemia
No full textThe defining characteristic of a stem cell is its capacity for extensive self-renewal and retention of multilineage differentiation potential (Gordon, 1993). The complex series of events required to guarantee self-renewal, proliferation and differ-entiation leads to the concept that the stem cell compartment has to be very heterogeneous and hierarchical (Ogawa, 1993). The transition from the primitive quiescent stem cell to the active functional hemopoietic cell requires various in-termediate stages characterised by the progres-sive loss of the self-renewal capacity and pro-gressive lineage restriction and commitment (Williams, 1993). This complex system is main-tained through the action of several regulatory molecules and signals originating from the he-mopoietic microenviroment. Up to now, a large number of growth factors, interleukins, and in-hibitory proteins have been identified, and their role in the regulation of the differentiation and proliferation processes have been extensively verified by many authors (Moore, 1991; Metcalf, 1993).
Several positive signals for proliferation and differentiation of stem/progenitor cells have been so far recognised, including the family of colony stimulating factors -CSF (GM-CSF, G-CSF, M-CSF, fibroblast-CSF, Meg-CSF, erythropoietin), interleukins (IL-1, IL-3, IL-6, IL-11, IL-12), and related molecules (Kit-ligand etc.) (Pistoia, 1992). However, the non proliferating state may be seen either as a passive process involving ab-sence of positive signals or an active process in-volving suppressive negative regulators that act as blocking, down modulating, or reducing the function of receptors for positive regulators on stem cells. Negative regulators may also inter-fere with signal transduction pathways, transcrip-tional factors, and mRNA production or stabil-ity, through the interaction with genes activated by positive regulators. They also act indirectly by blocking the synthesis of positive regulators. Some of these hemopoietic factors, such as TGF13 (transforming growth factor), TNFa (tumor necrosis factor), IFNs (interferons), and MIP (macrophage inflammatory protein) display pleiotropic activities
Flow cytometry measurement of cytokine receptors in acute leukemias. Clinical and biologic implications
No full textIn the last few years, the molecular cloning and purification of several hemopoietic growth factors (GF) and cytokines has made it possible to use them (at least some of these GF) in the clinical setting of many haematological disorders. The main clinical use of G- CSF and GM-CSF is to stimulate recovery of’ neutropenia and/or monocytopenia following myeloablative chemotherapy, radiotherapy, and/or bone marrow transplantation. Although the administration of these CF in patients affected by neoplastic proliferaiion of 1 lymphoid origin (ALL, MM, and MI.) is widely accepted, their clinical usefulness and safety in lhe management of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is still controversial. However, a number of reports have shown that CM-CSF anti G-NSF could be given to AML patients, without increasing the remission and the relapse rates or shortening the life expectancy of the disease. In othter words, the risk of stimulating the leukcrnic clone using GF seems to be very low even in patients with myeloid maligriancies
Flow cytometry measurement of GM-CSF receptors in acute leukemic blasts, and normal hemopoietic cells
No full textA quantitative analysis of expression levels of GM-CSF receptors was performed by flow cytometry in different disease categories, ie AML (n = 72), ALL (n = 18), and MDS (n = 12), as well as 12 healthy volunteers, using three different unconjugated GM-CSF/R monoclonal antibodies (McAbs) (HGM-CSFR (CD116), M5D12, 4B5F5), and appropriate standards. By using the reference HGM-CSFR McAb, in healthy subjects we found detectable levels of GM-CSF/R on blood monocytes (mean MESF (molecules of equivalent soluble fluorochrome)/cell: 36.1 x 10[3]), neutrophils (mean MESF/cell: 7.4 x 10[3]), bone marrow (BM) myelo-monocytic precursors (MESF range for the myeloid component, ie promyelocytes, myelocytes, metamyelocytes: 11.7-40.5 x 10[3], and for the monocytic lineage: 25.7-69.2 x 10[3]), and in two distinct subsets of BM CD34+ progenitor cells (GM-CSF/R dim: 2.5 x 10[3] MESF/cell, GM-CSF/R bright (10% of the total number of CD34 cells: 22.0 x 10[3] MESF/cell). In these subjects, there was no correlation between the expression levels of GM-CSF/R and CFU (CFU-GM, CFU-GEMM, BFU-E) colony production. Among the AML samples, M5D12 McAb was positive in 33%, 4B5F5 McAb in 90%, and HGM-CSF/R McAb in 78% of the cases examined (range of MESF/cell for the HGM-CSFR McAb: 0.9 x 10[3]-106.7 x 10[3]). The highest MESF values were seen in the M5 FAB subvariety (mean: 39.4 x 10[3]), where all the patients tested (n = 20) showed a strong positivity for the HGM-CSFR McAb. On the contrary, all ALL samples were GM-CSF/R negative except in two patients, who displayed a dim GM-CSF/R positivity (My+ALL: 1.3 x 10[3] MESF/cell; pro-B ALL: 1.0 x 10[3] MESF/cell). In most (>70%) M1 FAB subtypes, GM-CSF/R+ blasts co-expressed CD34low, HLA-DRhigh, CD33, CD38 antigens, and had little or no capacity to form CFU-GM colonies. GM-CSF/R+ blasts from the M5 FAB category were also positive for CD14, CD11c, CD33 and CD87. Furthermore, the number of GM-CSF/R expressed by leukemic cells from five out of 72 (7%) AML patients was above the highest values seen in normal samples (>69.2 x 10[3] MESF/cell), allowing the possibility of using this marker for the monitoring of the minimal residual disease (MRD) in a subset of AML. Cell culture studies aimed at evaluating GM-CSF receptor modulation following AML blast exposure to rhGM-CSF showed two distinct patterns of response; in the first group (6/10 cases) rhGM-CSF down-modulated GM-CSF receptors, whereas in the second group (4/10 cases), rhGM-CSF treatment was associated with either an increase or no change in the number of GM-CSF/R. In conclusion, cellular GM-CSF/R expression was variable and ranged from undetectable (ALL and a minority of AML) to very high intensities in M5 AML, and were also documented in some M0 AML, thus suggesting the concept that GM-CSF/R detection may be of help in lineage assignment of undifferentiated forms. Since the number of GM-CSF/R on AML blasts may be modulated after GM-CSF treatment, it can be postulated that the clinical use of GM-CSF in this disease may be optimized by a dynamic analysis of the number and the affinity status of GM-CSF-R in blasts and normal hemopoietic cells
CD116 (Granuclocyte-macrophage colony stimulating factor receptor)
No full textHuman CD116 is the alpha subunit of granulocytemacrophage colony stimulating factor receptor (GMCSFR, also called colony stimulating factor 2 receptor, alpha) that binds GM-CSF with low affinity. The molecule was termed CD116 at the 5th International
Workshop on Leukocyte Differentiation Antigens (IWLDA, Boston, USA, 1993). The beta subunit (CD131), which is also shared with the IL3 and IL5 receptors, has no detectable binding affinity for GM-CSF on it’s own but is necessary for high affinity binding when associated with the alpha subunit and plays a fundamental role in signal transduction.
Monoclonal antibodies (MoAbs) against CD116 (extracellular domain) are used for phenotyping various cell populations possibly contributing to the diagnosis and therapy of acute myeloid leukemia (AML).
In AML, GM-CSFR is detectable in 60-70% of cases and particularly in M4 and M5 FAB subvarieties. The number of receptors expressed by AML cells is sometimes significantly higher than that in normal hematopoietic cells, suggesting the possibility of using this marker as a useful tool for the monitoring of minimal residual disease. Cellular CD116 expression was documented in > 50% M0 AML.
Since the activity of GM-CSF on hemopoietic cells depends upon its binding to specific cell surface receptors, we have previously hypothesised that the clinical use of GM-CSF in AML patients could be optimized by a dynamic analysis of the number and the affinity status of GM-CSFR in leukemic blasts and normal hemopoietic cells
Flow cytometry evaluation of urokinase-type plasminogen activator receptor (UPA-R) in acute myeloid leukemia cells.
No full textThe aim of this study was to investigate by flow cytometry the expression of the UPA-R (Urokinase type plasminogen activator receptor-CD87) on the blastic population of AML and ALL patients in order to evaluate whether the presence of this molecule could be associated with peculiar clinical and biologic features of leukemic cells. Five different monoclonal antibodies (MoAbs) (clones: 3B10#; VIM5*; 109#; 68#; 100#) were used in order to detect the distinct forms of this cellular receptor. Cell reactivity varied significantly from case to case, also depending on the MoAb used for the flow cytometry analysis. In brief, 3B10# and VIM5* MoAbs were found to be positive in more than 90% of monocytes and neutrophils from healthy subjects, while the number of positive cells was decreased (60%) using the 109# MoAb. However, either 68# and 100# MoAbs recognised only a low number of blood monocytes and neutrophils (8-20%), while lymphocytes were unreactive with all the five UPA-R MoAbs. ALL cells were found to be CD87 negative in all cases. Blasts from AML showed a heterogeneous pattern of expression for the UPA-R MoAbs, being the reactivity strictly dependent on the MoAb used, and, to a higher extent, on the degree and type of maturation of the blastic cells. The number of blasts recognising 3B10# and VIM5* MoAbs was significantly higher than that reacting with the remaining MoAbs irrespective of the FAB subtype. Since proteolytic enzymes, like UPA, play a key role in the dissolution of the extracellular matrix, and in facilitating the cell egress from the bone marrow, it is conceivable that the expression of the UPA-R could contribute to the invasive properties and, possibly, metastatic potential of leukemic cells
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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