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Biomedical subjects

C C Stewart

Publications and source records attributed to C C Stewart.

At least 55 records · Page 3Linked to original sources
PubMed1995

Quantifying c-myc expression in c-myc antisense phosphorothioate oligodeoxynucleotide-treated leukemic and colon cancer cell lines.

Antisense oligodeoxynucleotides (oligo) have been used to inhibit oncogene expression and have potential therapeutic applications. Using a 15-mer antisense phosphorothioate oligo (S-oligo), inhibition of c-myc oncogene expression and cellular proliferation is studied in two cell lines with c-myc overexpression: a colon cancer cell line (Colo 320 DM) and a promyelocytic leukemic cell line(HL-60). Quantitative analysis of c-myc mRNA transcript is performed by competitive reverse transcription-polymerase chain reaction (RT-PCR). This utilizes an RNA competitive reference standard (CRS RNA) template that is identical to the native c-myc mRNA except for a short segment deletion to allow for differentiation of the two by gel electrophoresis. A fixed quantity of test mRNA and a series of known concentrations of CRS RNA template placed in the same test tubes under identical conditions are reverse transcribed and amplified by PCR. Since the reaction is competitive, the ratio of the PCR products reflects the ratio of the initial concentrations of the two templates. After gel electrophoresis, the two PCR products are quantified densitometrically. Treating Colo 320 DM and HL-60 cells with c-myc antisense oligo and S-oligo results in a 20- to 100-fold decrease in c-myc mRNA transcripts, respectively. This inhibition is dose dependent and sequence specific (c-myc sense and missense oligo have no effects). The quantitative decrease in c-myc mRNA is associated with corresponding inhibition of c-myc oncoprotein synthesis as demonstrated by flow cytometry and Western blots. Furthermore, there is inhibition of cellular proliferation of the respective cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Circulating natural killer cells but not cytotoxic T lymphocytes are reduced in patients with active relapsing multiple sclerosis and little clinical disability as compared to controls.

Triple antibody flow cytometry was used to compare the populations of CD56+ effector cells in the peripheral circulation of 29 patients with relapsing multiple sclerosis (MS) and little disability who were exacerbation-free for over 2 months and 29 healthy control subjects. Populations were characterized by two panels of antibodies (CD8, CD16, CD56 and CD3, CD8, CD56), as well as by size or granularity. In the MS patients, mature natural killer (NK) cells (CD3-CD8-CD56+) of small size and low granularity were significantly reduced compared to normals (P(0.0003). The quantities of other effector cells (cytotoxic T lymphocytes, large granular lymphocytes and monocytes) were not different in MS patients compared to the control subjects. Also, we identified a previously unrecognized population of CD56+ monocytes (CD3-CD14+CD56+) in both the normal control subjects and the MS patients which would have been misclassified as NK cells using one or two antibody cytometry employed in previous studies.

Antigens, CD↗
PubMed1995

Quantitative RT-PCR for measuring gene expression.

Classical Northern blot analysis for measuring mRNA requires too many cells to be practical for cell sorting. Yet, measurement of gene expression in small subsets within a heterogeneous population of cells is often desired. The PCR in combination with prior reverse transcription (RT-PCR) of the mRNA of interest provides a means for measuring gene expression using as few as one cell. When RT-PCR is performed, the reliability of the data can be highly subjective due to the efficiency of both RT and PCR steps. This subjectivity can be eliminated by a technique for quantitating specific RNA molecules using an internal RNA competitive reference standard (RNA-CRS), which is identical to the sequence of interest except for a deletion of 80 bases. Here we illustrate a strategy for quantitative PCR using a RNA-CRS, synthesized solely using nonplasmid-based PCR techniques. The competitive reaction consists of a constant quantity of wild-type mRNA (from 100-1000 cells) added individually to tubes containing a serially decreasing amount of RNA-CRS. The RT-PCR is performed on these samples, then the resulting product is analyzed by gel electrophoresis and densitometry. The procedure for preparing the RNA-CRS and subsequent RT-PCR steps are described in detail.

Animals↗
PubMed1994

Detection of HER-2/neu oncogene amplification in flow cytometry-sorted breast ductal cells by competitive polymerase chain reaction.

BACKGROUND: The amplification and/or overexpression of the HER-2/neu oncogene has been proposed as an important prognostic marker in breast cancer. However, contradictory results from various groups regarding whether there is statistical significance in HER-2 amplification or overexpression in predicting overall and disease free survival in node positive versus node negative patients exist in the literature. Current assays on quantifying the HER-2 oncogene rely on DNA extracted from homogenized breast tissue. Not only is a large amount of tissue required, but also, the DNA extract is contaminated with DNA from stromal cells and leukocytes, leading to decreased specificity and sensitivity of the HER-2 assay. Improving the specificity (DNA from breast ductal cells) and the sensitivity (competitive polymerase chain reaction [PCR]) of the HER-2 amplification detection assay will help resolve some of these controversies. METHODS: Using multiparameter flow cytometry (FCM), ductal cells from breast biopsies and fine needle aspirations (FNAs) are identified and selectively sorted using anti-cytokeratin, anti-HER-2 antibody labeling and DNA staining. HER-2 amplification in these sorted cells is then quantified by competitive DNA PCR using a competitive reference standard mutant template that is susceptible to the restriction enzyme Sma-1. RESULTS: Applying this strategy, SK-BR-3, an HER-2 amplified breast cancer cell line, was found to have approximately 9x baseline HER-2 oncogene copies. In addition, MCF-7, a known HER-2 nonamplified breast cancer cell line, was found to have baseline HER-2 oncogene copies. In the 10 clinical breast samples tested, 4 of the 10 breast cancers were HER-2 amplified using as few as 1000 cells. The cytokeratin positive cells of these cancers, in contrast to the cytokeratin negative cells, have detectably higher HER-2 amplification (7.2 +/- 2.8x versus 3.2 +/- 1.1x, respectively). Hence, HER-2 gene amplification would have been underestimated if unsorted cells were used because of stromal dilution. In the cytokeratin positive cells that were HER-2 oncogene amplified, corresponding HER-2 oncoprotein overexpression was detected by FCM. CONCLUSIONS: Using FCM, the ductal cell subpopulation of a breast specimen can be successfully sorted from breast biopsy and FNA specimens. Moreover, by applying the technique of competitive PCR, improved specificity and sensitivity in HER-2 oncogene amplification detection is achieved. The entire procedure can be accomplished in 1 day, allowing for a cost-effective assay and rapid turnaround time.

Base Sequence↗
PubMed1994

Multiparameter analysis of leukocytes by flow cytometry.

The flow cytometry described can be performed using a single laser. Each laboratory has to establish its own experience base and standard operating procedures. The intent of this discussion has been to illustrate the procedures that will lead to good flow cytometry data acquisition and analysis and to illustrate problematic areas. The most important rule of all is to recognize when there is a problem. It is hoped the information provided herein will be of help in the recognition process.

Cell Death↗
PubMed1994

A possible interpretation of some colony forming assays: 'developmental tree' model.

The characteristic form of the probability distributions of in vitro grown colonies is analysed and experimental results are shown to be well approximated by an exponential dependence. It is argued that this type of dependence is very frequent in nature and does not provide sufficient evidence to resolve the dilemma whether the development of haemopoietic lineages proceeds according to a schema of 'progressive and stochastic restriction' or more limiting 'developmental tree' with some random control process involved. Detailed analysis of the experimental conditions and results is needed to provide insight into collective phenomena in cellular proliferation and differentiation. At present, the complexity of the phenomena involved prevents unique interpretation of the experimental distributions. Nevertheless, one particular quantitative model, based on a developmental tree concept, yields a set of the theoretical probability distributions, which are in a very good agreement with the experimental results.

Animals↗
PubMed1993

Quantitation of c-myc gene amplification by a competitive PCR assay system.

Gene amplification is a common event in the progression of human cancers. The detection and quantitation of certain amplified oncogenes has been shown to have prognostic importance in certain human malignancies. A method is described that utilizes the principles of competitive PCR for quantitation of the c-myc gene copy number in relation to the copy number of a reference gene (tissue plasminogen activator [t-PA] gene) located on the same chromosome (8) as the c-myc gene. This ratio gives the true level of amplification of the c-myc gene, accounting for variables such as cell number, cell cycle phase, and chromosome 8 ploidy. The determination of gene amplification depends on the precise measurement of the ratio of target and reference genes. An important feature of this assay is that the competitive reference standards used for target gene c-myc and reference gene t-PA have been linked to form a hybrid. This simple modification guarantees that both reference gene and target gene assay tubes get identical amounts of the competitive template for each gene, thereby eliminating a significant source of error. This method has the same desirable attributes of standard PCR in that very small sample sizes are required and that results can easily be obtained in < 24 hr. In addition, this technique does not require the use of radioactivity or expensive DNA detection kits, and thus, may give it wider applicability for the study of human cancers.

Base Sequence↗
PubMed1992

Clinical applications of flow cytometry. Immunologic methods for measuring cell membrane and cytoplasmic antigens.

Flow cytometric analysis is the method of choice for classifying leukemia and lymphomas. When combined with other clinical and morphometric data, a definitive diagnosis can be made in the most difficult cases. By using a combination of specific antibodies that are each labeled with a different fluorochrome, panels can be designed to follow residual disease with greater precision than any other method. The detection of intracellular proteins is less well developed. Because antibodies can bind nonspecifically to dead cell components, there may be considerable biological noise. This noise reduces the sensitivity for detecting the desired protein within the cell. Until this problem is solved, it is likely that other methods may provide more reliable data. The major advantages of flow cytometry are rapid cell processing, cell by cell analysis, and the simultaneous evaluation of multiple markers. The latter is especially important because more tests can be performed using less clinical material. When flow cytometric results are combined with histologic results, the confidence in professional interpretation is far greater than either can provide alone. It is likely that the future application of these approaches will significantly improve diagnosis and patient care.

Antigens, Neoplasm↗
PubMed1992

Inhibitory role of interleukin-6 in macrophage proliferation.

We have shown that there are two forms of progenitor cells for macrophages. The first is characterized by a short lag period (about 1 day) before initiating the cell cycle, forms large colonies, is found in the bone marrow, and is in the nonadherent fraction. The second progenitor cell, found primarily in the adherent cell fraction of bone marrow and in peripheral tissues, forms small colonies after 14 days. We investigated the effect of combining interleukin-6 (IL-6) with colony-stimulating factor 1 (CSF-I) on macrophage proliferation. We found that IL-6 inhibited the proliferation of both types of progenitor cells, as well as more differentiated macrophages. This inhibitory effect was reversible because macrophages could initiate a proliferative response after removal of IL-6 from the culture medium. The introduction of anti-IL-6 into macrophage cultures containing IL-6 allowed proliferation, indicating that the effect was IL-6 specific. These results suggest that IL-6 may play a regulatory role in vivo by suppressing the production of bone marrow and tissue macrophages.

Bone Marrow Cells↗
PubMed1992

Effects of prestorage white cell-reduction of apheresis platelets on platelet glycoprotein Ib and von Willebrand factor.

Twenty plateletpheresis components were harvested from 11 healthy donors and stored in polyolefin bags on a horizontal flatbed agitator at 22 degrees C. After 24 hours, white cells were reduced in one aliquot by centrifugation while the other aliquot was stored unaltered. Samples were obtained aseptically from each of these platelets at intervals for up to 10 days, and measurements were made of platelet glycoprotein Ib (GPIb) by both flow cytometry and polyacrylamide gel electrophoresis, of ristocetin-induced platelet aggregation by impedance aggregometry, and of plasma and platelet von Willebrand factor (vWF) by enzyme-linked immunosorbent assay. Storage of platelets under these conditions was associated with only minor decreases in surface GPIb, intraplatelet vWF, and ristocetin-induced platelet aggregation, and no differences were observed between the white cell-reduced and nonreduced aliquots. No benefit of white cell reduction in such components before prolonged storage is evident in the vWF-platelet interaction.

Blood Component Removal↗
PubMed1992

Flow cytometric DNA analysis of excised breast lesions: use of fresh tissue needle aspirates obtained under guidance with mammography of the specimen.

Eighty consecutive biopsy specimens were studied to determine whether DNA analysis could be performed on fine-needle aspirates of excised clinically occult breast lesions obtained under guidance with mammography of the specimen before fixation to offer the advantages of fresh-tissue analysis. With use of single aspirates, cytologic analysis was possible in 50 cases (62%); DNA analysis was possible in 75 cases (94%). These methods combined offered no statistically significant increase in sensitivity for detection of malignancy compared with cytologic analysis alone. Forty-one percent of the analyzable invasive carcinomas showed aneuploidy. Aneuploidy and high S phase fractions of the invasive carcinomas showed no substantial correlation with patient age, nodal status, and size or appearance of the mammographic lesion. Aneuploidy was also seen in zero of four analyzable lesions showing ductal carcinoma in situ, two of 13 showing atypical hyperplasia (15%), and one of the 28 remaining benign lesions (4%). The authors conclude that this mammographic intervention is an effective means of obtaining fresh tissue samples of clinically occult lesions for DNA analysis.

Adult↗
PubMed1992

Functional characterization of mouse granulocytes and macrophages produced in vitro from bone marrow progenitors stimulated with interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF).

Bone marrow from C3H/ouj mice was depleted to < 1% of CD11b+ granulocytes and macrophages using paramagnetic beads coated with sheep anti-rat antibodies. CD11b- cells, enriched three- to fourfold in colony-forming cells, were stimulated in liquid culture with interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Cultures stimulated with IL-3 or GM-CSF increased cell numbers fourfold at 7 days, with the CD11b+ population increasing to 63% +/- 9% (n = 5) with IL-3 or 96% +/- 1% (n = 4) cells with GM-CSF. Functional responsiveness of the granulocytes and macrophages was assessed by flow cytometry in an oxidative burst assay using dichlorofluorescein (DCF) and a quantitative phagocytosis assay using opsonized fluorescent beads. Granulocytes and macrophages, identified by light scatter characteristics and allophycocyanine staining of CD11b, were assayed simultaneously with granulocytes from fresh mouse bone marrow and peripheral blood. GM-CSF-generated CD11b+ cells had higher oxidative responses than similar populations produced in response to IL-3. The oxidative burst of these in vitro generated CD11b+ populations was similar to the equivalent fresh bone marrow population. Oxidative burst responses of peripheral blood phagocytic cells could not be adequately measured in this system. Peripheral blood CD11b+ cells were the most phagocytic, followed by GM-CSF-stimulated CD11b+ cells; IL-3-stimulated and bone marrow CD11b+ cells were the least phagocytic. These data demonstrate that functional granulocytes can be produced in vitro using growth factors and that GM-CSF produces a more responsive cell than IL-3.

Animals↗
PubMed1992

Maximum-likelihood analysis of human T-cell X chromosome inactivation patterns: normal women versus carriers of X-linked severe combined immunodeficiency.

Lymphocytes of female carriers of X-linked severe combined immunodeficiency (XSCID; McKusick 300400; HGM genetic locus designation SCIDX1) exhibit nonrandom X chromosome inactivation. This phenomenon reflects a tissue-specific selective disadvantage for lymphocyte progenitors with an XSCID mutation on the active X chromosome and presumably is analogous to the process that inhibits T-cell development in affected boys with a single XSCID-bearing X chromosome. We investigated the specificity of T-cell X chromosome inactivation pattern as an indicator of immunodeficiency carrier status, as follows: X-inactivation ratios determined in a control group of noncarrier women exhibited a wide range, 20%-86% of T-cells with the paternal X active. Maximum-likelihood analysis of these data suggested that, in humans, mature T-cells are derived from a small pool of only about 10 randomly inactivated stem cells. Despite the wide variability in normal X-inactivation ratios, X inactivation in XSCID carriers appeared far more markedly skewed. Therefore a maximum-likelihood odds-ratio test was developed and proved to be successful in predicting the carrier status of women in XSCID pedigrees. This test has made it possible to identify XSCID carriers among mothers of boys with the heterogeneous syndrome of sporadic severe combined immunodeficiency.

Adolescent↗