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Biomedical subjects

C C Stewart

Publications and source records attributed to C C Stewart.

At least 19 recordsLinked to original sources
PubMed1998

Neutropenia associated with T-cell large granular lymphocyte leukemia: long-term response to cyclosporine therapy despite persistence of abnormal cells.

T-cell large granular lymphocyte (T-LGL) leukemia is clinically indolent, but is associated with severe neutropenia in approximately 50% of cases. The pathogenesis of the neutropenia is unclear. We report reversal of severe neutropenia associated with T-LGL leukemia in five patients treated with cyclosporine (CSA). All five had persistent neutrophil counts below 0.5 x 10(9)/L, two had agranulocytosis, and four had recurrent infections. Increased populations of LGL were present in blood and marrow, with a T-LGL immunophenotype (CD3(+)CD8(+)CD16(+/-)CD56(+/-)CD57(+)) shown by multiparameter flow cytometry, and clonal T-cell receptor (TCR) gene rearrangements in two of two pretreatment blood samples studied. CSA was initiated at doses of 1 to 1.5 mg/kg orally every 12 hours, with subsequent dose adjustments based on trough serum levels. Four patients attained normal neutrophil counts with CSA alone; one required addition of low-dose granulocyte-macrophage colony-stimulating factor. Time to attainment of 1.5 x 10(9)/L neutrophils ranged from 21 to 75 days. Attempts to taper and withdraw CSA resulted in recurrent neutropenia. Three patients have maintained normal neutrophil counts on continued CSA therapy for 2, 8, and 8.5 years. Two patients died 1.7 and 4.6 years after initiation of CSA despite normal neutrophil counts-one of metastatic melanoma and one of complications after aortofemoral bypass surgery. Despite resolution of neutropenia, increased populations of T-LGL cells have persisted in all patients during CSA therapy, as shown by morphology and flow cytometry and by the presence of clonal TCR gene rearrangements in four patients' posttreatment blood samples. We conclude that CSA is an effective therapy for neutropenia associated with T-LGL leukemia, and that resolution of neutropenia despite persistence of abnormal cells implies that CSA may inhibit T-LGL secretion of yet unidentified mediators of neutropenia.

Adult↗
PubMed1998

Direct isolation and analysis of endogenous transjunctional ADP from Cx43 transfected C6 glioma cells.

Gap junctional communication has been implicated in numerous cellular processes. However, the repertoire of specific transjunctional substances which mediate these processes remains relatively unexplored. A few selected secondary messengers have been identified, at least indirectly (e.g., cAMP and IP3) and phenotypic complementation experiments have indicated that gap junctions enable communicating cells to distribute nucleotide pools as a shared resource. The latter would include high energy compounds such as ADP and ATP, allowing cells to share energy resources. We have utilized a nonbiased process to directly capture, identify, and quantify transjunctional compounds from C6 glioma cells, the transformed phenotype of which has been ameliorated by transfection with connexin43 (Cx43). This technique involves the direct isolation, identification, and quantitation of radioactive transjunctional molecules that travel from metabolically labeled "donor" cells to "receiver" cells. This report demonstrates that ADP and/or ATP represents over 6% of the transjunctional material derived from glucose in Cx43-transfected C6 glioma cells. Furthermore, equilibration of these high energy metabolites among first order neighbors is shown to occur in less than 20 min of communication.

Adenosine Diphosphate↗
PubMed1998

Acute myeloid leukemia with 11q23 translocations: myelomonocytic immunophenotype by multiparameter flow cytometry.

11q23 translocations (t(11q23)) are recurring cytogenetic abnormalities in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia, involving the same gene, ALL1 (or MLL). Mixed lineage antigen expression has been reported in these leukemias, but its frequency and clinical significance are unknown. We immunophenotyped leukemia cells from 19 adult de novo AML patients with t(11q23) by multiparameter flow cytometry. Translocations included t(6;11)(q27;q23), t(9;11)(p22;q23), t(9;11;19)(p22;q23;q13.3), t(2;11)(11;17)(q37;q11q23;q11), t(11;17)(q23;q25), t(11;19)(q23;p13.1), t(11;19)(q23;p13.3) and t(11;22)(q23;q11). FAB types were M4 and M5. The committed stem cell and myeloid antigens HLADr, CD4dim, CD11b, CD13, CD15, CD32, CD33, CD38 and CD64 were each expressed in 80-100% of cases, and the early stem cell and lymphoid antigens CD34, CD56, CD3, CD2 and CD7 in 42, 39, 16, 5 and 5%, respectively. Antigen expression frequencies did not differ from those in 443 adequately karyotyped M4 and M5 cases without t(11q23). Fifteen patients (79%) attained complete remission (CR); median CR duration and survival were 10.0 and 15.1 months. CR duration and survival did not correlate with antigen expression. In particular, patients with t(9;11) survived longer than those with other t(11q23) (median not reached vs 7.6 months; P = 0.048), but antigen expression did not differ in the two groups. Thus frequencies of lymphoid antigen expression are similar in AML with t(11q23) and in other FAB M4 and M5 cases, treatment outcome does not differ in t(11q23) cases with and without lymphoid antigen expression, and better outcome of patients with t(9;11) compared to other t(11q23) does not correlate with differences in antigen expression. Mixed lineage antigen expression is not a distinctive feature of AML with t(11q23).

Adolescent↗
PubMed1998

Breast stereotactic core biopsy washings: abundant cell samples from clinically occult lesions for flow cytometric DNA analysis.

RATIONALE AND OBJECTIVES: The object of this study was to determine by flow cytometry the cellular and DNA content of the washings of core biopsy samples and needles obtained during stereotactic core biopsies of clinically occult suspicious lesions. METHODS: Fourteen-gauge core multipass biopsy inner-needle saline washings obtained after dislodgment of the tissue core samples (n = 30) and 14-ga mammotomy core biopsy specimen washings (n = 30) were analyzed by flow cytometry. RESULTS: Multipass core biopsy needle washings of 7 malignant and 23 benign lesions yielded a median of 10,584 cells per lesion (range 562-29,019 cells). Mammotomy core biopsy specimen washings of 10 malignant and 20 benign lesions yielded a median of 12,164 cells per lesion (range 1,295-115,150). Forty-four (73%) washings in each technique had more than 5000 cells. Flow cytometric DNA ploidy analysis was possible in 60 (100%) and the S phase percentage determination was possible in 59 (98%) of the washings. Aneuploidy was detected in 53% of malignant lesions and 2% of benign lesions. CONCLUSIONS: Core biopsy specimen or needle washings provide abundant fresh cellular material from clinically occult lesions for DNA analysis by flow cytometry.

Adult↗
PubMed1997

Expression of the neural cell adhesion molecule CD56 is associated with short remission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22).

Although acute myeloid leukemia (AML) with t(8;21) (q22;q22) is associated with a high complete remission (CR) rate and prolonged disease-free survival, treatment outcome is not universally favorable. Identifying factors that predict for treatment outcome might allow therapy to be optimized based on risk. AML with t(8;21) has a distinctive immunophenotype, characterized by expression of the myeloid and stem cell antigens CD13, CD15, CD34, and HLADr, and frequent expression of the B-cell antigen CD19 and the neural cell adhesion molecule CD56, a natural killer cell/stem cell antigen. Because CD56 expression has been associated with both extramedullary leukemia and multidrug resistance, we sought to correlate CD56 expression with treatment outcome in AML with t(8;21). Pretreatment leukemia cells from 29 adult de novo AML patients with t(8;21) treated on Cancer and Leukemia Group B (CALGB) protocols were immunophenotyped by multiparameter flow cytometry as part of a prospective immunophenotyping study of adult AML (CALGB 8361). CD56 was expressed in 16 cases (55%). There was no correlation between CD56 expression and age, sex, white blood cell count, granulocyte count, the presence of additional cytogenetic abnormalities, or the presence of extramedullary disease at diagnosis. The CR rate to standard-dose cytarabine and daunorubicin was similar for cases with and without CD56 expression (88% v 92%; P = 1.0). Post-CR therapy included at least one course of high-dose cytarabine in 24 of 26 patients who achieved CR; numbers of courses administered were similar in cases with and without CD56 expression. Although post-CR therapy did not differ, CR duration was significantly shorter in cases with CD56 expression compared with those without (median, 8.7 months v not reached; P = .01), as was survival (median, 16.5 months v not reached; P = .008). We conclude that CD56 expression in AML with t(8;21) is associated with significantly shorter CR duration and survival. Our results suggest that CD56 expression may be useful in stratifying therapy for this subtype of AML.

Acute Disease↗
PubMed1997

Specimen mammography-guided fine-needle aspirates of mammographically normal fatty and dense benign tissue: analysis of cell number and type.

Specimen mammography-guided 20-gauge fine-needle aspirates (FNA) were obtained from normal dense or fatty tissue. Single aspirates of dense tissue yielded greater cell counts; 74% (32 of 43) had greater than 5,000 cells as compared to 20% (19 of 93) of fatty tissue FNA, p < 0.05. Dense tissue FNA also yielded greater percentages of epithelial cells; 95% (21 of 22) had greater than 50% epithelial cells as compared to 43% (17 of 40) of fatty tissue FNA, p < 0.05. Mammographic guidance toward dense tissue is suggested for clinical studies of risk assessment using FNA of normal breast tissue.

Age Factors↗
PubMed1997

Specimen mammography-guided fine-needle aspirates of clinically occult benign and malignant lesions. Analysis of cell number and type.

RATIONALE AND OBJECTIVES: The authors determine the cell counts and percentages of epithelial cells in fine-needle aspirates (FNA) of mammographically detected breast lesions. METHODS: Specimen mammography-guided 20-gauge fine-needle aspirations were performed on 151 consecutive lesions. Cell counts were determined by flow cytometry of 106 consecutive aspirates. Semiquantitative determination of the percentage of epithelial cells was done by cytologic analysis of 151 aspirates. RESULTS: Single FNA cell counts were greater than 1000 for all lesions and greater than 7000 in 57% (31 of 54) of malignant and 35% (18 of 52) of benign lesions, P = 0.02. Fine-needle aspirates of soft tissue abnormalities had more than 7000 cells in 59% (27 of 46) of specimens, compared with 36% (22 of 61) in calcifications, P < 0.05. With the exception of fibroadenomas, the mammographic appearance of benign lesions, lesion size, and patient age had no association with cell counts. Ninety-three percent (76 of 82) of malignant lesion FNA and 80% (55 of 69) of benign lesion FNA had 50% or greater epithelial cells, P < 0.05. CONCLUSIONS: Fine-needle aspiration yields abundant cell counts of predominantly epithelial cells from most types of mammographically detected lesions and should be considered as a source of fresh cell samples for the study of benign and early malignant breast disease.

Adult↗
PubMed1996

CD34 progenitor cell subset analyses in normal human bone marrow and marrow harvested after intermediate-dose chemotherapy.

Previous attempts to characterize harvested marrow and peripheral blood stem cell (PBSC) in order to predict time to and quality of engraftment post autologous bone marrow transplant (autoBMT) have included use of in vitro colony forming unit (CFU) assays. These assays are hampered by interlaboratory variability and are not uniformly predictive. CD34 quantification by flow cytometric technique has also been used to assess the quality of harvested marrow and PBSC. However, a lack of standardization has hampered direct comparison of published reports. We sought to characterize these early lineage-committed CD34+ progenitor cells from non-ficolled harvested marrow with six progenitor cell (PC) panels containing CD34 antibody plus two additional early lineage markers, using multiparameter flow cytometry. The specific gating technique including simultaneous CD34-PE vs. side scatter and forward vs. side scatter, was verified using morphologic analyses of sorted CD34+ cells. An ungated file was initially acquired to assess total CD34+ content. A second file using a CD34 threshold was then acquired to resolve lineage-committed subsets. The % CD34+ cells as well as cells/microliter of bone marrow was calculated using cell counts at the time of marrow harvest. Bone marrow (mean total cell dose = 3.8 x 10(5)/kg), obtained from 42 normal donors for allogeneic transplantation was first analyzed. CD34+ cells comprised a mean 1.3% of non-ficolled marrow, with 328 CD34+ cells/microliter, and mean CD34+ cells collected was 4.8 x 10(6)/kg. While no significant differences in total cells harvested nor proportion of CD34+ cells was found, a significant decrease in CD34 cells/microliter (= 233, P = .0012) was found in cancer patients. The percentage of CD19+ and CD38+ progenitor cells was significantly increased, while CD5+ and CD71+ cells were decreased. The proportions of all other early lineage-committed CD34 subsets were not different. Measurement of lineage-committed CD34 progenitor cells is a useful technique to characterize harvested marrow and PBSC, and may be applied to predict time and quality of engraftment post ablative conditioning regimens.

Adult↗
PubMed1996

Invasive breast carcinoma: analysis of dynamic magnetic resonance imaging enhancement features and cell proliferative activity determined by DNA S-phase percentage.

BACKGROUND: There is little information regarding associations between magnetic resonance imaging (MRI) enhancement and biologic parameters of breast carcinoma. A prospective study was undertaken to correlate MRI dynamic contrast enhancement features with cell proliferative activity, as determined by DNA S-phase percentage. METHODS: Seventeen patients with invasive breast cancer underwent MRI at 1.5 tesla using a dynamic gadolinium-enhanced spoiled gradient recall echo technique. DNA analysis of samples of the excised lesions was then performed using flow cytometry. RESULTS: Invasive carcinomas with high DNA S-phase percentages (> or = 6.9%, the median value in this study), a measure of increased cell proliferation, were associated with a peripheral MRI enhancement pattern in 4 of 6 (67%) lesions compared with 0 of 11 carcinomas with lower DNA S-phase percentages (< or = 6.9%) (P = 0.006). There was no significant association between a high DNA S-phase percentage and greater MRI enhancement amplitude, rate, or washout. There was no significant association between aneuploid DNA content and any MRI enhancement feature. CONCLUSIONS: Increased cell proliferation in invasive breast carcinoma, as determined by high DNA S-phase percentage, is significantly associated with a peripheral MRI enhancement pattern but unrelated to greater MRI enhancement amplitude, rate, or washout.

Adult↗
PubMed1996

Cellular proliferative activity of mammographic normal dense and fatty tissue determined by DNA S phase percentage.

A prospective study was undertaken to evaluate associations among mammographic normal dense and fatty tissue, benign histologic findings, and cellular proliferative activity determined by DNA S phase percentages. Mammographic and histologic findings of benign, normal dense and fatty tissue contained in 100 excised specimens were cross-tabulated with flow cytometric DNA S phase percentages of needle aspirates obtained under radiographic guidance. Histologic evidence of atypia or hyperplasia was present in 21 (32%) of 66 fatty tissue samples and 11 (32%) of 34 dense tissue samples (p = NS). The median S phase percentages were identical for mammographic fatty and dense tissue (median % S phase = 5.35). The frequency of high S phase percentages (above the median) was not significantly different for tissue containing atypia or hyperplasia, 47% (15 of 32), as compared to tissue containing other benign histology, 52% (35 of 68) (p = NS). These frequencies were similar in women 49 and younger or 50 and older. These data show that mammographic normal dense and fatty tissues contain similar frequencies of histologic atypia or hyperplasia and low and high cellular proliferative activity determined by DNA S phase percentages in women aged 49 and younger and 50 and older.

Adipose Tissue↗
PubMed1995

Prolonged administration of low-dose interleukin-2 in human immunodeficiency virus-associated malignancy results in selective expansion of innate immune effectors without significant clinical toxicity.

Ten adult patients with human immunodeficiency virus (HIV)-associated malignancies (five with lymphoma and five with Kaposi's Sarcoma) were treated with a daily subcutaneous injection of interleukin-2 (IL-2) for 90 consecutive days in a phase I dose-escalation study. Seven patients had absolute CD4 counts below 200/mm3 at the time malignancy was diagnosed. Each lymphoma patient had obtained a complete or partial remission with standard chemotherapy before initiating IL-2. The daily dose of IL-2 did not change during the 90-day course of therapy. Seventeen courses of IL-2 therapy were completed at doses ranging from 0.4 x 10(6) U/m2/d to 1.2 x 10(6) U/m2/d without significant (grade III) toxicity. Two of two patients experienced grade III toxicity within 21 days of initiating IL-2 at a dose of 1.4 x 10(6) U/m2/d, but both patients subsequently completed 90 days of therapy at the maximum tolerated dose (MTD) of 1.2 x 10(6) U/m2/d. Although there were no significant increases or decreases in T-cell subsets at any dose level, there was an increase in absolute natural killer (NK) cell number at the three highest doses of IL-2 (mean percent increase 247; 95% confidence interval, 124 to 369) that was statistically significant (Wilcoxon one-sample signed rank test, P = .015). One patient developed an anti-IL-2 antibody titer that correlated with minimal NK cell expansion in vitro and in vivo. An increase in eosinophils was noted during 9 of 17 courses of IL-2 therapy without correlation to IL-2 dose, prior course of IL-2, or NK cell expansion. At the MTD, there was no consistent increase in the plasma HIV RNA level over time. Three of 10 patients had progressive disease while on study. During 50 months of IL-2 therapy, no patient was treated for an opportunistic infection. We conclude that daily low dose subcutaneous IL-2 can be self-administered safely with good compliance for prolonged periods of time to patients with HIV-associated malignancies, including those with profound immune deficiency. The majority of patients show selective expansion of innate immune effectors, ie, NK cells and/or eosinophils, in the absence of significant clinical toxicity or increased viral burden. These results suggest that low-dose IL-2 therapy should be studied further in phase II clinical trials for evidence of activity against malignancy and opportunistic infection in this patient population.

Adult↗
PubMed1995

Amplification and detection of a Y-chromosome DNA sequence by fluorescence in situ polymerase chain reaction and flow cytometry using cells in suspension.

A procedure for amplifying and detecting nucleic acid sequences in situ using cells in suspension and flow cytometry has been developed. The process involves the use of the polymerase chain reaction (PCR) and a fluorescent in situ hybridization (FISH) protocol developed in our laboratory to detect the amplified PCR product. For these studies, a Y-chromosome specific repeat DNA sequence was amplified. Daudi cells, a B-cell lymphoma culture line established from a male, was used as a positive control and HL-60, a promyelocytic leukemia culture line established from a female, was used as a negative control. During the in situ PCR process cellular autofluorescence (noise) increases causing markedly reduced detection sensitivity of the probe (signal) bound to the amplified product within the positive cells. An autofluorescence reduction circuit was applied which was integrated into as standard bench top flow cytometer to reduce this noise, thereby producing a 10-fold increase in detection sensitivity of the signal. Without the application of the autofluorescence reduction circuit, the positive control histogram distribution was virtually indistinguishable from the negative control sample distributions. After autofluorescence reduction, the data showed that the Y-chromosome DNA was only amplified in the Daudi cells subjected to the complete in situ PCR protocol. This increased sensitivity also provided direct detection of the Y-chromosome repeat sequence, albeit exhibiting less signal compared to the amplified target after the in situ PCR.

Base Sequence↗
PubMed1995

Classification and regression trees for bone marrow immunophenotyping.

Methods are needed to assist with automating three-color flow cytometric immunophenotyping of bone marrow from leukemia patients. Described is a method in which a normal bone marrow data set is used as a template against which to compare leukemic bone marrow data sets. This template is obtained using techniques of cluster analysis and cluster editing. Leukemic cells often inappropriately express antigens and appear in a different part of the multivariate data space than normal cells. To recognize the cells exhibiting inappropriate antigen expression, an artificial cluster of "cells" is added to the normal template. The "cells" in this cluster fill the space not occupied by normal cells. Classification and regression tree (CART) analysis is used to train a classifier that can then be used to isolate the major cell types and the inappropriate expression cells in a leukemic bone marrow specimen.

Automation↗
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↗