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M G Pallavicini

Publications and source records attributed to M G Pallavicini.

At least 19 recordsLinked to original sources
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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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PubMed1987

Quantitative flow cytometric and clonogenic evaluation of glass bead affinity fractionation of antibody-labeled murine bone marrow.

Monoclonal antibodies and a glass bead affinity fractionation technique were used to selectively deplete subpopulations of murine bone marrow cells. Flow cytometric analyses permitted quantitative measurement of subpopulation depletion and characterization (light scatter and fluorescence intensity) of both the eluted and bound cell subpopulations. Mouse bone marrow cells were labeled with selected monoclonal rat anti-mouse antibodies directed against cell surface antigens and were eluted through a glass bead column coated with goat anti-rat immunoglobulin. Unlabeled cells passed through the column, whereas cells labeled with the antibody were selectively retained. Column operating conditions for optimal depletion of labeled cells were determined. With specific column conditions, 97% of the antibody positive cells were retained on the column. In addition, clonogenic assays on cells sorted from unfractionated and column fractionated preparations provided estimates of the fraction of granulocyte macrophage progenitors (CFU-GM) in the different cell subpopulations. The enrichment of CFU-GM achieved in the eluted cell populations was dependent upon the antibody used for cell labeling and ranged from four- to six-fold. Since large numbers of cells can be processed rapidly, this technique, in combination with antibodies specific for non-clonogenic cells, is particularly suitable when preparations enriched in colony-forming progenitors are required.

Animals↗
PubMed1987

Multivariate flow cytometric characterization and enrichment of murine megakaryocyte progenitors (CFU-Meg).

Multivariate flow cytometric analysis and sorting of mouse bone marrow cells viably stained with Hoechst 33342 (HO) was utilized to obtain a subpopulation enriched at least 30- to 60-fold for clonogenic megakaryocyte precursors (CFU-Meg). HO fluorescence intensity, forward light scatter, and perpendicular light scatter were measured on each HO-stained bone marrow cell as it passed through a dual beam flow cytometer. Cells were sorted on the basis of their light scattering and HO fluorescence intensity properties. The sorting region which included cells with high forward light scatter, low perpendicular light scatter, and low (0.1-0.65 times the modal channel) HO fluorescence intensity contained approximately 0.6% CFU-Meg. Of all CFU-Meg, 75% were contained in the light scatter gate, and 56% of all CFU-Meg were in the low HO-sorting region. This technique offers a simple, one-step procedure to obtain preparations enriched in murine clonogenic megakaryocyte precursors.

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PubMed1986

Cell cycle analysis using flow cytometry.

This manuscript reviews the utility of flow cytometry for the study of cell proliferation. The applications of univariate DNA distribution analysis to cytokinetic studies of asynchronous and perturbed cell populations are discussed briefly. The newly developed technique for simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated bromodeoxyuridine is discussed in more detail. The cytochemistry required for this analysis is reviewed as are its applications to: determination of the fractions of cells in the G1-, S- and G2 + M-phases of the cell cycle; determination of the G1-, S- and G2 + M phase durations and dispersions and growth fraction for asynchronous cells; detection of ara-C resistant cells present at low frequency in an otherwise sensitive population; and analysis of the cytokinetic response of a solid murine tumour to treatment in vivo with a cell cycle specific agent.

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PubMed1985

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity.

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.

Animals↗
PubMed1985

Multivariate analysis and list mode processing of murine hemopoietic subpopulations for cytokinetic studies.

Multivariate analyses and list mode data processing were used to obtain cytokinetic information on flow cytometrically distinct hemopoietic subpopulations. In one application, viable, unfixed hemopoietic subpopulations were discriminated on the basis of cyanine dye fluorescence and orthogonal light scatter; Hoechst dye fluorescence was measured to determine the proliferative status of the subpopulations. In another application, ethanol-fixed mouse bone marrow cells were triply stained with Hoechst dye, rhodamine-conjugated wheat germ agglutinin (WGA), and a fluorescein-labeled monoclonal antibody against bromodeoxyuridine. In both of these studies, flow cytometric data for all three variables were acquired in list mode fashion, stored on magnetic tape, and processed by list mode software on a computer-based multivariable pulse-height analyzer. In the first application, subpopulations distinguished by cyanine dye intensity and light scatter appeared to be more related to cell lineage and cell size than proliferative status. In the second application, WGA affinity discriminated two subpopulations in mouse bone marrow S-phase cells in each subpopulation that actively incorporated bromodeoxyuridine (BrdUrd). List mode data processing obviates the need for routine electronic sorting of cells and thus facilitates characterization of discriminated subpopulations. In this regard, it is particularly useful for the study of flow cytometrically distinct, low frequency subpopulations.

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PubMed1985

Cytokinetic properties of asynchronous and cytosine arabinoside perturbed murine tumors measured by simultaneous bromodeoxyuridine/DNA analyses.

This paper describes the determination of cytokinetic properties of asynchronous and cytosine arabinoside- (Ara-C) treated KHT tumors growing in vivo using the bromodeoxyuridine (BrdUrd)/DNA analysis technique. The cytokinetic properties of asynchronously growing tumors were estimated by computer analysis of sequential BrdUrd/DNA distributions measured at 2- to 3-h intervals after administration of a single i.p. injection of BrdUrd. The cytokinetic properties of the Ara-C-treated tumors were estimated by computer analysis of BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment. BrdUrd was injected 30 min prior to tumor harvest. The cytokinetic properties of the cells rendered nonclonogenic by Ara-C were followed in BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment of tumors that were labeled with BrdUrd 30 min prior to Ara C injection. The G1-, S-, and G2M-phase durations were estimated to be 7.6, 10.9, and 2.0 h prior to Ara-C; decreasing to 1.2, 4.1, and 1.4 after Ara-C. The growth fraction was estimated to be 0.8 prior to Ara-C. Complete recruitment of the normally noncycling subpopulation was observed after Ara-C treatment. Ara-C-killed cells were removed from the tumor within 24 h following Ara-C injection. These cytokinetic properties were similar to those reported in other studies.

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