Assessment of distribution of CD34 epitope classes in fresh and cryopreserved peripheral blood progenitor cells and acute myeloid leukemic blasts.

So far several reports have described changes in the expression of surface antigens in progenitor cells and blasts following cryopreservation. However, there are no data on the effects of cryopreservation on the expression of the three CD34 epitope classes, and on their relationship with the clonogenic capacity of PBPC collected by leukapheresis. DESIGN AND METHODS: In order to analyze the effects of freezing/thawing procedures (Eth 80C storage for 3 months) and use of dimethylsulfoxide (DMSO) on the immunophenotype profile and colony production of peripheral blood progenitor cells (PBPC) in apheresis products derived from 20 patients with stage 0-III non-Hodgkin's lymphoma (nHL), a flow cytometry study was undertaken using different CD34 monoclonal antibodies (MoAbs) capable of recognizing the 3 epitope classes of CD34 molecule (class III: HPCA-2/FITC, HPCA-2/PE, 581/FITC, 581/PE; class II: Q-Bend 10/PE; class I: ICH3/PE, BI3C5-PE, Immu-133-PE). CD34 epitope expression was also analyzed in thawed CD34+ blasts obtained from 14 patients with acute myeloid leukemia (AML), who were analyzed using a larger number (#17) of CD34 epitope class I, II, and III reactive MoAbs. RESULTS: Under our experimental conditions it was found that class III and class II CD34 epitopes (differentially resistant to enzymatic cleavage with neuraminidase, chymopapain and glycoprotease) are better preserved than class I epitope Eth sensitive to degradation Eth after cell exposure to cryoprotectant DMSO and the freezing- thawing procedures. Results further showed a concomitant decrease in class I CD34+ counts and in BFU-E colony production. A significant increase in CD34 antigen expression levels (i.e. antibody binding capacity, ABC) by cryopreserved cells stained with CD34 epitope class III, and class II reactive MoAbs was also documented, while no changes after cryopreservation were noted using class I-reactive MoAbs. The slight increase in the percentage of CD34+ cells detected after frozen storage was correlated to a concomitant decrease in the number of more mature myeloid cells (CD15+, CD13+, CD33+). Compared to pre-cryopreservation values, a slight reduction in class I CD34 epitope expression was also found in thawed CD34+ AML blasts. INTERPRETATION AND CONCLUSIONS: As far as the reduction of class I CD34 epitope is concerned, it may be hypothesized that the freezing procedure, use of DMSO, and/or lysis methodology may either damage a CD34 subset, or induce distinct alterations of the CD34 glycoprotein, possibly determining a reduction in their immunoreactivity with some CD34 MoAbs. In conclusion, this study has shown that exposure to the cryoprotectant DMSO and the freezing/thawing procedures modifies the distribution of CD34 epitopes as well as the clonogenic capacity of PBPCs from nHL patients, and CD34+ blasts from AML. These findings need to considered when selecting CD34 MoAbs for enumeration and positive selection of stem/progenitor cells for research and clinical purposes

Micosi profonde

Le micosi profonde sono infezioni che interessano solitamente il polmone, le quali possono eventualmente disseminare per via ematica con coinvolgimento degli organi interni e della cute (micosi disseminate o sistemiche) con intensa reazione immunitaria da parte dell’ospite. Sono causate, sia da funghi patogeni presenti nell’ambiente che da funghi opportunisti. I funghi patogeni causano malattie sia negli ospiti immunocompetenti che in quelli immunocompromessi. La loro frequenza è notevolmente aumentata in seguito al diffondersi dell'infezione da HIV/AIDS. In questo capitolo vengono descritte la candidosi, l'aspergillosi, la criptococcosi, l'istoplasmosi, la coccidioidomicosi, la blastomicosi nord-americana, la paracoccidioidomicosi (blastomicosi sud-americana), la zigomicosi, l'infezione da Penicillium marneffei ed infine la pneumocistosi, grave infezione polmonare sostenuta da Pneumocystis jirovecii

Educazione all'igiene orale e prevalenza di Rothia dentocariosa in un campione di popolazione infantile. Risultati di un'indagine condotta nella città di Ferrara

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CD116 (GM-CSF-R)

Human 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

Immunophenotype definition of the stem/ progenitor cell compartment. Implications for the identification of blast cells in acute leukemia

The 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

Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic MPO and lysozyme in normal and leukemic cells

Using 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