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R Miglierina

Publications and source records attributed to R Miglierina.

17 recordsLinked to original sources
PubMed1991

A simple diagnostic test for Fanconi anemia by flow cytometry.

A simple diagnostic test for Fanconi anemia (FA) by flow cytometry is proposed. It is based on the cell cycle disturbances of FA cells and their sensitisation by alkylating agents. Following PHA-stimulation of whole blood cell cultures in the presence or absence of nitrogen mustard, the accumulation of cells in G2/M phase was measured. A sharp increase of cells in G2/M was observed in cultures from FA patients when nitrogen mustard was added. This increase allows one to distinguish FA patients from patients with anemias of other origin, healthy controls, and FA heterozygotes, as effectively as chromosome breakage studies. The rapidity of the test and its reliability as demonstrated on the ten FA patients studied, will make the diagnosis of FA easier in centers without cytogenetic laboratory facilities.

Cell Cycle↗
PubMed1990

Diagnosis of Fanconi's anemia by flow cytometry.

FA is a progressive bone marrow aplasia genetically transmitted by a recessive autosomal gene or genes. In our laboratory, cytogenetic diagnosis is based on evaluation of the chromosomal breakage of mitotic cell derived from patient blood-cell cultures and sensitized by nitrogen mustard (NM). We have observed, in parallel with this test, fluctuations of the cell cycle of PHA- stimulated peripheral blood lymphocytes from FA patients as compared with controls. FA cells treated with NM show a dramatic and significant increase in G2/M phase after 72 hr in vitro culture, compared with untreated or control cells (normal controls and non-FA patients). This test is rapid and simple, as it consists in staining cells with a DNA dye (propidium iodide), followed by a flow cytometry analysis of the cell cycle phases. Our results in twelve patients are correlated with the cytogenetic results.

Cell Cycle↗
PubMed1990

Lymphocyte activation via MHC class II antigens.

Class II antigens of the major histocompatibility complex (MHC) have a clear role in restricting cellular interactions and presenting processed antigen to T cells. In addition, a fundamental role for class II antigens in cellular activation has been suggested, following studies which demonstrated that class II antigen binding altered signal transduction in various cell types. We have investigated the role of HLA class II antigens in signal transduction of human B lymphocytes. Resting B lymphocytes proliferate in response to immobilized anti-class II antibodies and intracellular free calcium is increased following cross-linking of anti-class II antibodies. The two dimensional PAGE pattern of resting B lymphocytes was examined and differences were noted after stimulation with either anti-class II antibodies, anti-IgM or PMA. The 2D PAGE pattern following stimulation with anti-class II antibodies was not identical to that observed following stimulation with either anti-IgM or PMA. These data suggest that the activation of B-Go via the class II antigens shares part of the pathway of anti-IgM induced activation but does not follow an identical pathway.

B-Lymphocytes↗
PubMed1989

Different Hoechst 33342 and DAPI fluorescence of the human Y chromosome in bivariate flow karyotypes.

The staining properties of AT-specific dyes Hoechst 33342 and DAPI as revealed by Hoechst 33342/mithramycin and mithramycin/DAPI bivariate human flow karyotype patterns are different for chromosomes rich in heterochromatin. The peak corresponding to chromosome Y of a given cell line is higher on the A/T axis with mithramycin/DAPI staining than with mithramycin/Hoechst. The chromosome 1 was found slightly more fluorescent in mithramycin/DAPI than in mithramycin/Hoechst as judged by the slight displacement of its area on the Hoechst-DAPI axis. The other peaks did not show major differences. On the same flow karyotypes, chromosomal rearrangements were detected.

Benzimidazoles↗
PubMed1989

Chromosome mapping of the human ras-related rab3A gene to 19p13.2.

The rab genes belong to one of the three main branches of the ras super family. The encoded rab proteins share 38 to 75% amino acid identity with the yeast YPT1 and SEC4 proteins. We used the human rab3A cDNA to map the corresponding gene on human chromosomes by chromosome sorting and in situ hybridization. Both techniques allowed the assignment of the rab3A gene to chromosome 19 with a regional localization on 19p13.2 obtained by in situ hybridization.

Chromosome Banding↗
PubMed1988

Single-cell analysis of the intracellular pH and its regulation during the monocytic differentiation of U937 human leukemic cells.

Monocytic differentiation of U937 cells induced by retinoic acid is accompanied by a 0.2-pH-unit cell alkalinisation. The effect of retinoic acid on intracellular pH (pHi) develops slowly and it precedes the differentiation of the cells by 24 h. Heterogeneity in cellular pHi values was assessed using flow cytometry. It was higher at the differentiated stage than at the undifferentiated stage. It was reduced under conditions of clamped pHi values. Two membrane mechanisms allow U937 cells to recover from an intracellular acidosis. These are the Na+/H+ exchange system and a Na+-dependent HCO3-/Cl- exchange system. The increase in the pHi observed after monocytic differentiation resulted from a twofold increase in the maximum activity of the Na+/H+ exchange system with no change in the activity of the bicarbonate-dependent system. The properties of interaction of the Na+/H+ exchanger of U937 cells with Na+, Li+, amiloride and its derivatives were defined and appeared to be unique to human leukemic cells.

Amiloride↗
PubMed1988

Cross-linking of membrane IgM on B CLL cells: dissociation between intracellular free Ca2+ mobilization and cell proliferation.

It has been shown previously that chronic lymphocytic leukemia (CLL) B cells are frozen at different stages of activation with unique requirements for proliferation. Although most B CLL cells express surface IgM, anti-mu antibodies are able to trigger only some of them to proliferate and/or respond to B cell growth factor (BCGF) or interleukin 2 (IL2), as normal B cells. In this report we extend these observations using three different monoclonal antibodies (mAb) to human mu chain (one mitogenic in soluble form for normal B cells, the two others mitogenic only when coupled on Sepharose 4B beads). Cells from only 3 out of 11 B CLL patients proliferated in the presence of either mitogenic anti-mu. When the early events following surface Ig cross-linking, such as calcium mobilization (by flow cytometry on indo-1-labeled cells), were studied all three mAb in soluble form were able to induce a similar increase in the intracellular free calcium concentration ([Ca2+]i); analogous to [Ca2+]i rise after the mitogenic F(ab')2 anti-mu stimulation. This response was seen only in 8 out of the 12 CLL B cells studied. All B CLL cells, however, proliferate in response to a combination of phorbol ester 12,13-dibutyrate (PBt2) and ionomycin. Therefore, three patterns of response to sIg cross-linking by anti-mu could be distinguished: cells from 4 out of 12 cases proliferate and mobilize Ca2+ upon anti-mu triggering (behaving like resting B lymphocytes); in 4 other cases anti-mu lead to Ca2+ mobilization without cell proliferation; in the last 4 cases neither Ca2+ mobilization, IP3 generation (in the one case studied) nor cell proliferation are observed although these cells do proliferate directly in response to growth factors. Moreover, anti-mu stimulation in this group leads to increased proliferation in response to BCGF and IL2 suggesting an anti-Ig signaling independent of inositol phosphate metabolism. These results are interpreted in terms of differential anti-mu signaling on B cells at different stages of activation.

Antibodies, Anti-Idiotypic↗
PubMed1988

The ras-related ral gene maps to chromosome 7p15-22.

Human cDNAs coding for the new protein ral that shares 50% homology with the ras proteins have been recently isolated. A 600-bp fragment carrying mainly the coding region was used to localize the ral gene by hybridization with sorted chromosomes and in situ hybridization. Direct molecular hybridization on sorted chromosomes using a single laser illumination allowed the assignment of the ral gene to a region of the flow karyotype containing chromosomes 7, 8 and X. With dual laser analysis ral was assigned to the fraction containing chromosome 7. In the 331 human metaphases hybridized with the 3H-labelled insert, the silver grain distribution showed a unique major signal on chromosome 7p15-22.

Cell Line, Transformed↗
PubMed1986

Immunological typing of acute lymphoblastic leukemia: concurrent analysis by flow cytofluorometry and immunocytology.

For 60 cases of acute lymphoblastic leukemia (ALL) immunological typing was done concurrently by the avidin-biotin-peroxidase method using cytocentrifuged smears and by flow cytofluorometry for the study of surface antigens. The use of a large panel of antibodies detecting differentiation antigens allowed us to sub-classify 57/60 cases as 43 B-lineage ALLs and 14 T-lineage ALLs. The two types of ALL can be accurately distinguished by the expression of the antigens recognized by the antibodies of the clusters of differentiation CD19 (B4) and CD7 (Leu 9). Almost perfect agreement was obtained between the results of the two methods for antigens DR, CD10 (cALLA;J5) and CD7. A number of discordances were observed with other antigens [CD19 (B4), CD20 (B1), CD22 (To15), CD1 (T6), CD2 (T11), CD4 (T4), CD8 (T8), CD3 (T3), T9, T10]. In spite of these discordances, the avidin-biotin-peroxidase method can predict the lineage involved in most ALLs with a high degree of reliability. Nevertheless, for weakly expressed surface antigens (such as B4 and B1) the immunocytological method is less sensitive than flow cytofluorometry and can only approximately determine the stage of differentiation of neoplastic cells. Furthermore, the existence of cases which are at the same time negative with flow cytofluorometry and positive with immunocytology is consistent with the intracytoplasmic expression of certain differentiation antigens. Thus in the course of lymphoid differentiation, intra-cytoplasmic expression of T3, To15 and possibly J5 precedes their expression at the cell surface.

Antigens, Surface↗
PubMed1986

Some applications of microspheres in flow cytometry.

Microspheres of latex ranging from 0.1 to 10 microns in diameter are nowadays commercially available. Labelled with different fluorochromes, they are good standards to check the optical path and to calibrate flow cytometers. The kits available contain microspheres highly selected with a precise size and a narrow distribution, so as to allow an easy alignment of instruments. Mixed with the cell preparations, microspheres may be used as an internal standard, in order to compare samples from day to day. Phagocytic activity has been investigated after incubation of cells with fluorescent microspheres. Covalently bound to ligands such as antibodies, microspheres become a powerful and specific reagent to label cell surface antigens. The positive signal is enhanced, allowing the detection of poorly represented epitopes. This paper reviews some examples of these applications published in the flow cytometry field.

Animals↗
PubMed1986

Flow cytogenetics.

Classical cytogenetics is often tedious and many efforts have been made to develop other methods of chromosome analysis, among which flow karyotyping has recently emerged. Although less efficient than banding techniques to identify each chromosome, flow cytometry offers the opportunity of analyzing large quantities of chromosomes at a very high rate, resulting in a flow karyotype. Even if the initial aim of this technique, namely clinical diagnosis, has not been reached, another major application has emerged, namely chromosome sorting. This method is unique for isolating a set of purified chromosomes from most cells grown in culture in sufficient amount to perform experiments using molecular biology techniques. Significant results have been already obtained either through the construction of chromosome-specific libraries or in the assignment of DNA probes to particular sorted chromosomes.

Animals↗
PubMed1986

A monoclonal antibody against an erythrocyte ontogenic antigen identifies fetal and adult erythroid progenitors.

A murine monoclonal antibody (MoAb) designated FA6-152 has been obtained by immunizing mice with fetal erythrocytes. This antibody agglutinates fetal but not adult erythrocytes. Among blood cells, this antibody bound to both adult and fetal monocytes, platelets, and reticulocytes, but did not react with lymphocytes and granulocytes. Fluorescent labeling of marrow cells and of in vitro BFU-E, CFU-GM, and CFU-MK-derived colonies has shown that the antigen defined by FA6-152 MoAb was absent from the granulocytic precursors and was detected on the megakaryocytic lineage at a later stage of differentiation than the platelet-specific markers. In contrast, the antigen appeared as a very early marker of the erythroid differentiation since all erythroblasts, including proerythroblasts, were labeled even before the expression of glycophorin A. Cells from adult marrow and fetal liver were sorted with the FA6-152 MoAb and studied by electron microscopy and cell culture. The negative fraction contained granulocytic, monocytic, and megakaryocytic precursors, whereas the positive fraction was devoid of these precursors and contained monocytes, erythroblasts at all stages of maturation, and a homogeneous population of blasts. Cultures have shown that the only hematopoietic progenitors present in this positive fraction were CFU-E and some BFU-E. The antigenic density was related to the differentiation stage of the erythroid progenitors. In conclusion, this antibody is similar to the previously described 5F1 MoAb (Bernstein and Andrews, J Immunol 128:876, 1982; and Andrews et al, Blood 62:124, 1983) and provides a useful probe for studies leading to improved understanding of normal and malignant erythroid differentiation.

Animals↗
PubMedDate not supplied

Immunochemical study of a human myeloma IgG1 half molecule.

The serum of a patient with multiple myeloma contained an IgG1 kappa monoclonal protein which existed in two molecular species: one with and one without inter-heavy chain covalent bonds, the latter dissociating into half molecules without reduction. The half molecules were present in the urine together with a kappa Bence-Jones protein. This peculiar protein was discovered because the serum and urinary IgG formed double percipitin lines when analysed by immunoelectrophoresis with an antiserum to gamma chains, the inner line being due to residual normal IgG. The isolated half molecule, as well as the major constituent of the 7 S IgG fraction, failed to precipitate with most antisera specific for the Fc fragment of IgG. The half molecule lacked the Gm(non a) marker and other antigenic determinants of the third constant region of gamma1 chains. No isotypic or allotypic antigenic determinant of another immunoglobulin class or subclass was detected. The molecular weights of the 7 S molecule, the half molecule and its covalently linked heavy and light chains were about normal, suggesting that they did not have a large deletion which could have caused the lack of interheavy chain covalent bonds. The hinge peptide appeared normal after high voltage electrophoresis of the peptic-tryptic digest of the reduced and alkylated half molecule. The carboxy-terminus of the heavy chain and the amino acid composition of the molecule were similar to those of IgG1 myeloma proteins. Two cysteinyl peptides of the CH3 domain showed on a diagonal peptidic map an electrophoretic mobility somewhat different from that of the corresponding peptides of an IgG1 myeloma protein. Another peculiar feature of this protein was the presence of galactosamine. Idiotypic determinants of the half molecule were present in the 7 S fraction, suggesting that the 7 S IgG molecules and the half molecules were derived from the same clone of tumour cells. Lack of material precluded further characterization of the structural abnormality--probably located in the third constant domain of the heavy chain--responsible for the absence of most antigenic determinants of the Fc region of IgG1 and for the formation of half molecules. This abnormality could be a small deletion undetectable by molecular weight measurements or an unidentified exchange of genetic material. Family members of the patient could not be studied in order to investigate whether this immunoglobulin abnormality reflected a minor genetic variant or a mutational event.

Amino Acid Sequence↗