PubMed Health⌕ Search

Biomedical subjects

M G Pallavicini

Publications and source records attributed to M G Pallavicini.

At least 19 recordsLinked to original sources
PubMed1995

Cytogenetically aberrant cells in the stem cell compartment (CD34+lin-) in acute myeloid leukemia.

Leukemia may be viewed as a clonal expansion of blast cells; however, the role of primitive cells and/or stem cells in disease etiology and progression is unclear. We investigated stem cell involvement in leukemia using fluorescence in situ hybridization (FISH), immunofluorescence labeling of hematopoietic subpopulations, and flow cytometric analysis/sorting to discriminate and quantify cytogenetically aberrant stem cells in 12 acute myeloid leukemia (AML) and three myelodysplastic (MDS) specimens. Flow cytometric analysis and sorting were used to discriminate and collect a primitive subpopulation enriched in stem cells expressing CD34+ and lacking CD33 and CD38 (CD34+lin-). A subpopulation containing progenitors and differentiating myeloid cells expressed CD34, CD33, and CD38 (CD34+lin+). Nine specimens contained less than 10% CD34+ cells and, thus, were considered to be CD34- leukemias. Mature lymphoid, myeloid, and erythroid subpopulations were sorted on the basis of antigen-linked immunofluorescence. Cytogenetically aberrant cells in sorted subpopulations were identified using FISH with enumerator probes selected on the basis of diagnosis karyotype. Cytogenetically aberrant CD34+lin- cells were present at frequencies between 9% and 99% in all specimens. CD34+lin- cytogenetically aberrant cells comprised between 0.05% and 11.9% of the marrow/blood specimens. Cytogenetically aberrant CD34+lin+ cells constituted 0.01% tp 56% of the marrow/blood population. These data demonstrate that aberrant cells are present in primitive CD34+ stem cell compartments, even in CD34- leukemias. Stem cell involvement was confirmed further by sorting lymphoid and erythroid subpopulations from eight specimens in which the predominant leukemic population lacked lymphoid/erythroid differentiation markers. In these specimens, as well as in multiple lineages, suggests involvement of a cell(s) with multilineage capabilities. The ability of aberrant CD34+lin- stem cells to contribute to clonal and compartment expansion within immunofluorescently defined subpopulations was evaluated to explore the functional phenotype of aberrant CD34+lin- cells. Analysis of compartment size and aberrant cell frequency suggests that frequency of cytogenetically aberrant stem cells is uncoupled from compartment size. These data suggest that cytogenetically aberrant cells in the primitive compartment show varying abilities to expand primitive compartments. Cytogenetically aberrant CD34+lin- cells precede the blast subpopulation in hierarchical maturation and may in some cases by considered preleukemic, requiring maturation or additional mutations before transformation (eg, compartmental expansion) occurs.

ADP-ribosyl Cyclase↗
PubMed1995

Unusual kinetics of white cell clearance in transfused mice.

BACKGROUND: Donor white cells (WBCs) in blood transfusions are responsible for complications in recipients, including alloimmunization, graft-versus-host disease (GVHD), and virus transmission and reactivation. The recent use of sequence-specific polymerase chain reaction assays to monitor the kinetics of clearance of donor WBCs in transfused humans and dogs found transient recirculation of donor lymphocytes on Days 3 to 5 after transfusion; this presumably reflected an abortive GVHD reaction to major histocompatibility complex-incompatible recipient cells, after which donor WBCs were cleared to undetectable levels. STUDY DESIGN AND METHODS: This study sought to develop a murine model to further characterize the kinetics and major histocompatibility complex restriction of donor WBC clearance. A sensitive murine Y chromosome-specific polymerase chain reaction assay was developed and applied to serial blood samples collected after transfusions of allogeneic blood to naive inbred, primed inbred, and outbred mice, as well as after transfusions of gamma-radiated blood to naive inbred mice. RESULTS: In inbred mice, both naive and primed to the allogeneic blood donor, transfused WBCs were not cleared to undetectable levels for more than 1 month after transfusion. Transfused outbred mice also showed prolonged donor WBC survival, although at lower levels than inbred mice. There was no evidence of GVHD in either inbred or outbred mice, and gamma radiation had no significant impact on donor WBC persistence. CONCLUSION: These results contrast with the rapid clearance of donor WBCs observed in humans and dogs. The immunologic basis for this discrepancy remains unclear. Caution should be exercised in any extrapolation to humans of conclusions drawn from results in transfused mice.

Animals↗
PubMed1994

Intracellular dynamics of c-myc mRNA traffic in single cells in situ.

Processing and intracellular transport of RNA transcripts are essential for gene expression. Translational regulation of gene expression may occur by several mechanisms, including control of transcript movement from sites of synthesis to site(s) of translation. We describe temporal analysis of the intracellular translocation of c-myc transcripts from the site of synthesis in the nucleus to sites of translation in the cytoplasm. Fluorescence in situ hybridization (FISH) and quantitative fluorescence microscopy were used to measure intracellular traffic of c-myc transcripts in individual recombinant cells following activation of c-myc sequences linked to a heat shock promoter. C-myc nuclear transcripts are visible in the nucleus within minutes of heat exposure. Transcripts remain in the nucleus for at least 4 hr after gene activation. Transport of c-myc transcripts to the cytoplasm begins approximately 1 hr after cells are returned to 37 degrees C. These data demonstrate the feasibility of measuring intracellular transcript transport following gene activation and provide a description of the kinetics of intracellular traffic of inducible c-myc transcripts in heated cells in situ.

Animals↗
PubMed1994

A mouse chromosome 11 library generated from sorted chromosomes using linker-adapter polymerase chain reaction.

We describe the generation of a mouse whole chromosome library using sequence-independent polymerase chain reaction (PCR) to amplify sequences contained in DNA extracted from flow sorted chromosomes. DNA in sorted chromosomes from a human x mouse hybrid cell line was digested with a frequent four-cutter restriction enzyme, Sau3AI, and the ends were ligated to an adapter oligonucleotide. The ligated DNA fragments were amplified using PCR primers homologous to the linker-adapter oligonucleotide. PCR-generated products were characterized by gel electrophoresis. The size of the amplified DNA ranged from 100 to more than 1,000 bp with a relatively high proportion of products at approximately 400 bp. Biotinylated PCR products used for FISH showed specific hybridization to murine metaphases and no hybridization to human lymphocyte and hamster metaphase cells. Banding analysis indicated that the probes were specific for mouse Chromosome 11. We anticipate that availability of painting probes for specific murine chromosomes will facilitate cytogenetic studies in the mouse.

Animals↗
PubMed1993

Molecular cytogenetics: solid tumors and leukemia.

Acute leukemia represents one of the best-studied malignancies. Consequently, numerous dogmas have evolved over the years. One dogma to which we subscribe is that leukemia is a genetic disease and that the behavior of leukemic cells and their response to therapy is determined largely by their genetic characteristics. The relative ease of metaphase generation and culture of single leukemic cells, well-defined morphologic criteria, and availability of phenotyping reagents has contributed to a wealth of information about the karyotypic, genetic, and functional properties of these cells. Nevertheless, establishment of genotype-phenotype relationships has been particularly elusive. The genetic evolution of leukemias and the consequences of specific aberrations remains to be determined. We describe the power of new molecular cytogenetic tools, fluorescence in situ hybridization, and comparative genomic hybridization to measure the genetic evolution of leukemic cells. Lessons learned from application of these techniques to solid tumors and speculations about similar approaches to analysis of leukemia are addressed.

Cytogenetics↗
PubMed1993

Competitive repopulation in leukemic and normal bone marrow.

The success of chemotherapy in leukemias in which the marrow appears entirely replaced by leukemic cells must be due to the persistence of some normal stem cells. The implied competition between leukemic and normal stem cells is thus akin to the competition between donor and host cells in irradiated animals. A review of that competition points to the importance of the quantitative relationships between the competing stem cells, even when one of the competing stem cell clones has a proliferative advantage. Pursuing that analogy, it is suggested that stimulating the surviving normal stem cells by appropriate combinations of cytokines may be of therapeutic benefit, once the tumor load has been reduced by chemotherapy. Complete eradication of leukemic cells may not be necessary, if surviving normal cells could gain ascendancy over the residual leukemic cell clones.

Animals↗
PubMed1992

Comparison of strategies to detect and quantitate uniquely marked cells in intra- and inter-species hemopoietic chimeras.

Evaluation of the outcome of successful bone marrow transplantation and indepth studies of transplantation biology rely increasingly upon detection and enumeration of donor hemopoietic cells in the transplanted recipients. The ability to detect and enumerate low levels of donor engraftment in interphase cell subpopulations in hemopoietic chimeras is particularly important for studies of mixed lineage chimerism, early relapse manifestations, and engraftment of subpopulations present at low frequency. We describe and compare the sensitivity and specificity of DNA-based detection strategies (fluorescence in situ hybridization, in vitro DNA amplification using the polymerase chain reaction) and flow cytometric analysis of cell surface markers to detect cells carrying marker DNA or proteins in syngeneic (mouse-to-mouse) and xenogeneic (mouse-to-human, monkey, sheep) backgrounds. DNA-based detection strategies offer advantages of rapid analysis and enumeration of target cell frequencies with detection sensitivities approximating 10(-4). The sensitivity of immunofluorescence-linked flow cytometric-based detection of nucleated leukocytes approached 10(-3), whereas flow cytometric-based detection of fixed human erythrocytes was feasible at cell frequencies of 10(-5). Data described in this manuscript should facilitate selection of appropriate methodologies for assessment of hemopoietic chimerism following transplantation.

Animals↗
PubMed1992

Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero.

Hemopoietic stem cells from human fetal liver were transplanted in utero into preimmune fetal sheep (48-54 days of gestation). The fate of donor cells was followed using karyotype analysis, by immunofluorescence labeling with anti-CD antibodies, and by fluorescent in situ hybridization using human-specific DNA probes. Engraftment occurred in 13 of 33 recipients. Of five live born sheep that exhibited chimerism, all expressed human cells in the marrow, whereas three expressed them in blood as well. Engraftment was multilineage (erythroid, myeloid, and lymphoid) and human hemopoietic progenitors (multipotent colony-forming units, colony-forming units-granulocyte, macrophage, and erythroid burst-forming units) capable of forming colonies in vitro were detected in all five lambs for greater than 2 yr. These progenitors responded to human-specific growth factors both in vitro and in vivo. Thus the administration of recombinant human IL-3 and granulocyte macrophage-colony-stimulating factor to chimeric sheep resulted in a 2.1-3.4-fold increase in the relative expression of donor (human) cells. These results demonstrate that the permissive environment of the preimmune fetal sheep provides suitable conditions for the engraftment and long-term multilineage expression of human hemopoietic stem cells in a large animal model. In this model, donor human cells appear to retain certain phenotypic and functional characteristics that can be used to manipulate the size of donor cell pool.

Animals↗
PubMed1992

Integration and stability of CHO amplicons containing plasmid sequences.

Fluorescence in situ hybridization (FISH, 15) and high density, non-selective long term perfusion culture were used to study aspects of genetic stability and productivity in recombinant CHO cells. We analysed the distribution and structure of recombinant amplicons in chromosomes of CHO cells used for the production of proteins. In the presence, but not in the absence, of methotrexate (MTX) we found a high proportion of cells (40-60%) with multiple and/or unusually structured and extended chromosome regions containing amplified sequences. Removal of MTX from culture media resulted in the rapid decline in the frequency of cells containing amplified sequences exhibiting multiple and heterogeneous integrations. In cloned lines cultivated in the absence of MTX, a well defined signal motif on a specific chromosome, interpreted as the "master integration" unit, became increasingly abundant over time until almost all cells contained that signal motif. We used fluorescence in situ hybridization (FISH) with chemically modified DNA probes complementary to the integrated sequences to verify the stability of master integrations in cells cultured for extended periods in medium lacking MTX. In a second approach, we studied long-term stability of recombinant sequences during non-selective perfusion culture of CHO populations with non-cloned, MTX resistant heterogeneous populations of cells producing CD4IgG chimeric molecules. Due to the mode of transfection and primary selection the resulting cell lines consisted of subpopulations of cells derived from various independent integration events. The amplification and MTX selection procedure used consecutively generated multiple, structurally different amplicon types in the cell population, thus increasing the degree of heterogeneity. High density perfusion culture of these cells in the absence of MTX was used to maximize growth rates and was thought to select against cells with reduced growth rates, due to the highly amplified state of their introduced DNA, with concomitantly high productivity. However we found no evidence for such a selection; cells showed no reduction in copy number or total loss of amplified sequences at the end of the culture. More significantly, specific productivity of these cell lines grown under non-selective conditions did not decrease over the 100 days observation period.

Animals↗
PubMed1991

Transgene integration in hair follicles and peripheral blood cells measured by in vitro DNA amplification and fluorescence in situ hybridization.

Screening of animals to detect the presence of integrated DNA sequences is an essential component of transgenic mouse generation. Rapid and sensitive detection techniques to facilitate identification of transgenic animals for biological studies or subsequent breeding programs are desirable. Most transgenics are generated on F1 backgrounds, thus determination of the histocompatibility status of neonates provides important diagnostic information for establishing congenic colonies. We describe the application of two assays, in vitro DNA amplification using the polymerase chain reaction (PCR) and fluorescence in situ hybridization with biotinylated DNA probes, to facilitate rapid detection of transgenes and their chromosomal integration patterns in young mice. A noninvasive PCR-based assay to detect the transgene in DNA contained in detergent-extracted hair follicles was developed for rapid screening. A total of 147 mice derived from F2, F3, and F4 generations of C57BL x F1 (globin transgenics) were assayed to determine whether they carried a globin transgene. Characterization of animals by PCR-based amplification of the transgene was compared with that obtained using standard Southern analysis of DNA extracted from tails. Categorization of animals as positive (carrying the transgene) or negative using PCR was performed successfully in the initial assay with 95% of the animals. Fluorescence in situ hybridization with a DNA probe showing homology with a portion of the transgene was performed on metaphase and interphase cells to determine the integration pattern of the transgene. Our data showed that the transgene was integrated in a single chromosome. These techniques should facilitate rapid identification of transgenic animals and characterization of the genomic transgene integration patterns.

Animals↗
PubMed1991

Successful stable xenograft of human fetal hemopoietic cells in preimmune fetal sheep.

Human fetal liver hemopoietic stem cells were transplanted into preimmune fetal sheep. Significant numbers of recipient fetuses showed evidence of engraftment of human stem cells which responded in vivo to human-specific hemopoietic growth factors. The chimerism has persisted now for > 2 years without significant graft loss. No evidence of GVHD has been noted. Successful engraftment was associated with the expression of human erythroid, myeloid, and lymphoid differentiation. This xenograft model offers useful possibilities for the study of the biology of human hemopoiesis in vivo.

Animals↗
PubMed1991

Short- and long-term repopulation of lethally irradiated mice by bone marrow stem cells enriched on the basis of light scatter and Hoechst 33342 fluorescence.

Murine bone marrow subpopulations enriched in hemopoietic stem cells were transfused into lethally irradiated hosts to determine the contribution of host cells and two types of donor cells to marrow repopulation. Donor cell suspensions were a mixture of marrows from two congenic lines of mice containing electrophoretically distinguishable alloenzymes of phosphoglycerate kinase (PGK-A and PGK-B). The donor cells were sorted by high forward light scatter, low-to-intermediate perpendicular light scatter, and low Hoechst 33342 fluorescence intensity. The congenic hosts contained a third distinct marker, glucose phosphate isomerase (GPI-A). The two markers in the donor cells allowed determination of the clones generated by the seeded cells over a 36-week period of observation. The clone number declined rapidly during the first 12 weeks following transplantation and reached stable levels at 20 weeks, indicating the number of long-term repopulating cells (LTRC). The sorted subpopulation was enriched 170-fold for day-13 spleen colony-forming units (CFU-S), 235-fold for cells providing a 30-day survival, and 136- to 160-fold for LTRC. Survival for the 36-week observation period was 40%-100% for groups of hosts receiving 100-3000 sorted cells and 80% for controls receiving 2 x 10(5) unsorted cells. In all groups, similar distribution of phenotypes among peripheral blood erythrocytes, platelets, and lymphocytes at 36 weeks suggested that the repopulating donor stem cells were pluripotential. Transfusion of 3000 sorted cells, containing about 5 LTRC and 60 CFU-S, assured continuous repopulation with 95%-100% donor cells 4 to 36 weeks after transplantation, whereas significant numbers of host cells re-emerged temporarily or permanently when lower numbers of LTRC and CFU-S were transfused. The data indicate that both the quality and quantity of pluripotential stem cells in sorted bone marrow are important for complete long-term marrow reconstitution.

Animals↗
PubMed1990

Simultaneous flow cytometric detection of cellular c-myc protein, incorporated bromodeoxyuridine, and DNA.

We describe a multivariate flow cytometric technique for simultaneous analysis of specific nuclear protein, bromodeoxyuridine (BrdUrd) incorporated into DNA and DNA content in single cells in suspension. The procedure involves fixation of BrdUrd-exposed cells with paraformaldehyde, heat denaturation of cellular DNA, followed by sequential immunochemical reactions to label incorporated BrdUrd and nuclear protein, and finally staining of total DNA with propidium iodide. The cells are analyzed flow cytometrically and multivariate data acquired in list mode to facilitate analyses of heterogeneous subpopulations. We applied this technique to measure c-myc protein, incorporated BrdUrd, and DNA content in subpopulations present in a recombinant Chinese hamster ovary (CHO) cell line carrying approximately 800 copies of murine c-myc sequences under control of an inducible heat shock promoter.

Animals↗
PubMed1990

Relationship of c-myc gene copy number and gene expression: cellular effects of elevated c-myc protein.

The relationship between gene copy number and expression and cellular consequences of elevated levels of c-myc protein has been investigated using recombinant Chinese hamster ovary cell lines transfected with DNA coding for the murine c-myc gene. HC-8 and LC-5 recombinant cells carry approximately 800 and 50 copies of c-myc sequences, respectively, under control of an inducible heat shock promoter. Multivariate flow cytometric analysis and clonogenic assays were used to measure the relationship among c-myc expression, rate of DNA synthesis, and cell survival. Following heat exposure, maximally induced HC-8 cells produced approximately tenfold more c-myc protein than heated LC-5 cells, suggesting a close relationship between gene copy number and level of expression. However, considerable heterogeneity in the level and time of c-myc expression was observed following heat induction, even though the amounts of genomic c-myc were relatively constant. Heterogeneity in gene expression was not attributable to variation in heat induction methodologies and/or cell cycle phase distributions. The presence of high levels of recombinant c-myc protein was associated with a decreased rate of bromodeoxyuridine incorporation into DNA. High levels of c-myc protein in HC-8 cells were inversely correlated with cell survival postheating, suggesting that high levels of c-myc protein are incompatible with cell survival.

Animals↗
PubMed1990

Effects of methotrexate on transfected DNA stability in mammalian cells.

The chromosomal locations, amounts, and level of expression of transfected, amplified c-myc and dihydrofolate reductase sequences were measured in cells cultured in the presence and absence of methotrexate. These studies show that the location and amount of transfected sequences, as well as the level of expression, were more variable when the cells were cultured in methotrexate.

Animals↗
PubMed1989

Rapid screening and selection of monoclonal antibodies by bivariate flow cytometric analyses.

We describe a bivariate flow cytometric assay to rapidly identify hybridomas producing new monoclonal antibodies recognizing subpopulations that are unreactive with existing immunological reagents. In this screen, whole cells in microtiter wells are labeled first with a red-linked test antibody, and then with a green-linked cocktail of existing monoclonal antibody reagents. The multiply-stained fluorescent cells are analyzed flow cytometrically and bivariate distributions of red vs. green-linked antibody fluorescence are generated. Test antibodies that recognize different subpopulations than those labeled by antibodies in the cocktail are readily identified. The use of an antibody cocktail conjugated with a single fluorophore allows comparison of the reactivity of the test antibody with multiple existing antibodies in a single analysis. This screen allows rapid (approximately 100 test antibodies can be evaluated in 40 min) identification of potentially interesting new antibodies for discrimination of subpopulations in heterogeneous tissues. We describe application of this assay to identify antibodies useful to mark hemopoietic subpopulations.

Animals↗
PubMed1989

Use of fluorescence in situ hybridization to detect and monitor transfected and amplified sequences in recombinant CHO cells.

Non-radioactive fluorescence in situ hybridization was used to detect transfected and amplified sequences in recombinant CHO cells. The cell lines used in this study are based on DHFR-deficient CHO-DUKX cells. The recombinant CHO cells contain and express, as verified by Southern and Northern experiments, multiple copies of a constitutive DHFR expression vector, as well as an inducible Drosophila HSP 70 promoter-mouse c-myc construction. In order to localize and monitor the chromosomal location of transfected and amplified DHFR and c-myc sequences, biotinylated DNA probes were hybridized to metaphase preparations of several cell lines. The resulting hybrids were detected using fluorescein-linked avidin. The fluorescence signal was amplified using a biotinylated anti-avidin antibody. The number of c-myc and DHFR integration sites per metaphase, their distribution in cell populations growing at various methotrexate levels, the sizes of the amplified sequences, as well as the number of chromosomal rearrangements were measured. The results of this study will be presented and the usefulness of this method as a general tool for rapid characterization and monitoring of recombinant cell lines will be discussed.

Animals↗