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Biomedical subjects

J P McCoy

Publications and source records attributed to J P McCoy.

At least 19 recordsLinked to original sources
PubMed1998

Complete replication of human sperm genome in egg extracts from Xenopus laevis.

To examine the ability of Xenopus egg extracts to support a complete replication cycle of human sperm genome, demembranated human spermatozoa were incubated with the extract from activated Xenopus laevis eggs. Most sperm heads were decondensed within 15 min. The heads became round within 30 min with diameters of 10-30 microns. The process of DNA replication in the pronuclei was monitored by two methods, bromodeoxyuridine incorporation and flow cytometry. The results indicate that DNA replication was initiated approximately 1.5 h after membrane structure formation and that it lasted up to 9 h. The amounts of DNA in most pronuclei were doubled by 4-9 h, depending on which donor toad was the source of the egg extract. Inclusion of the protein synthesis inhibitor, cycloheximide (100 micrograms/ml), had no obvious effect on human sperm DNA replication but appeared to prevent the pronuclei from degradation after a prolonged period (> 6 h) of incubation. After storage in liquid nitrogen for > 1.5 mo, the efficiency of the egg extracts in supporting sperm head decondensation and DNA replication was reduced for human sperm but not for Xenopus sperm. Possible applications of the use of Xenopus egg extract for human sperm activation and DNA replication are discussed.

Animals↗
PubMed1996

Immunophenotypic analysis of the T cell receptor V beta repertoire in CD4+ and CD8+ lymphocytes from normal peripheral blood.

The antigenic specificity of the majority of T lymphocytes in the peripheral blood is determined by the combination of alpha and beta variable region chains present in the T cell receptor complex. Currently, the V beta chains are grouped into 25 families. Historically, determination of V beta usage has relied on detection of gene rearrangement on the nucleic acid level; however, with the increased availability of monoclonal antibodies to the product of these genomic rearrangements, immunophenotypic methods are rapidly becoming a reliable alternative method for studying the usage of V beta regions by T cells and T cell subsets. In the present study, multiparametric flow cytometry was used to determine the use of 10 V beta chains on CD4+ and CD8+ T cells in the peripheral blood of 28 normal donors. By obtaining absolute lymphocyte counts at the time blood was drawn, the absolute number of both CD4+ and CD8+ cells using particular V beta regions could be determined. Additionally, the intradonor consistency of V beta usage was examined by obtaining blood from 5 of the volunteers at an interval of approximately 1 year. The results of this study suggest a fairly homogeneous pattern of use for these V beta regions. The most striking longitudinal differences were observed in one individual who underwent a tonsillectomy midway between the T cell receptor V beta determinations.

CD4-Positive T-Lymphocytes↗
PubMed1996

Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis.

Determination of DNA ploidy has been found to be of diagnostic and prognostic value with regard to many solid tumors. Flow cytometric analysis of DNA ploidy is dependent on the binding of fluorescent dyes to DNA. Preserving cell morphology by fixing the tissue in formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA. This distortion of DNA content measurement can cause inaccuracies in DNA-ploidy determinations of formalin-fixed tissue specimens and precludes the use of appropriate DNA standards. Therefore, it has been impossible to determine accurately the DNA ploidy of formalin-fixed, paraffin-embedded (FFPE) tissues. Using formalin-fixed cells as a model for FFPE cells, we developed a thermal treatment method to reverse the effect of formalin on the binding of propidium iodide to DNA. Applying this approach to the preparation of FFPE lymph node and breast tissue for DNA analysis, we have developed a method that makes the binding of PI to the DNA of FFPE tissue mimic that of fresh tissue. Following dewaxing, rehydration, and trypsin treatment, the FFPE tissue, resuspended in PBS, was heated to 75 degrees C for 90 min to restore the PI binding to that of fresh cells. This method makes it possible to use fresh, DNA-diploid cells as an internal control and, thus, determine more accurately the DNA ploidy of tumors preserved in formalin and paraffin.

Aneuploidy↗
PubMed1996

A survey of current practices in clinical flow cytometry.

In mid-1995, a 40-question survey was distributed to determine the current practices of clinical flow cytometry laboratories. Forty-seven responses were received. Included in the survey were questions regarding the affiliation and size of the laboratory, the qualifications of the staff, the nature and number of assays performed, whether laboratory resources and specimen loads were changing, and how data were analyzed and interpreted. The results indicate considerable variability in many aspects of clinical cytometry, such as number of markers used for immunophenotyping leukemias and lymphomas, types of specimens analyzed for DNA-ploidy, and charges for specific tests.

Cytodiagnosis↗
PubMed1995

Immunophenotyping of congenital leukemia.

Congenital leukemia is a rare but well-documented disease in which a leukemic process is detected at birth or very shortly thereafter. An estimated 175-200 reports of congenital leukemia have appeared in the literature. The majority of the cases reported have not undergone thorough immunophenotyping, but rather have been assigned lineage based on cytochemical and morphological studies. Historically, a large proportion of congenital leukemias have been thought to be of the myeloid lineage, in contrast to pediatric leukemias in general, which are primarily lymphoid. The precise proportions of the lineage assignments may be distorted by the inclusion of cases of transient myeloproliferative disorders (TMD) as congenital leukemia. The immunophenotyping data available to date suggest that congenital leukemias are phenotypically heterogeneous, lacking any common distinguishing markers. The prognosis for congenital leukemias is usually poor if leukemoid reactive processes, such as TMD, are carefully excluded.

Cell Lineage↗
PubMed1995

Congenital leukemia: report of two cases.

Congenital leukemia is a rare disease in which a leukemic process is present at birth or immediately thereafter. The majority of cases presented in the literature were reported prior to the availability of contemporary immunophenotyping methods, and lineage assignment was often made on the basis of morphology alone. Congenital leukemias may be of various lineages, although, historically, monocytic and myelomonocytic congenital leukemias appear to be the most prevalent. We present two cases of congenital leukemia with detailed immunophenotypic and cytochemical characterization. One case is of the lymphoid lineage, and the second is of myelomonocytic lineage. Neither patient displayed trisomy 21.

Female↗
PubMed1995

Alterations of T-cell receptor variable region expression in human immunodeficiency virus disease.

Since only a small percentage of CD4+ lymphocytes is infected at any one time during the course of human immunodeficiency virus (HIV) disease, a question central to the pathogenesis of HIV is whether or not the depletion of CD4+ lymphocytes is a random or selective event. The majority of peripheral blood T lymphocytes use alpha and beta variable chains as components of their T-cell receptor (TCR) complex. Depletion of CD4+ T lymphocytes from the peripheral blood may be dependent on the V beta chain expressed by the CD4+ cell, based on the hypothesis that HIV may encode a superantigen. Peripheral blood from normal controls and HIV+ patients was studied for alterations in the expression of various V beta chains of the TCR. Three-color flow cytometry was used to determine the expression of V beta 2, V beta 3, V beta 8, V beta 13, and V beta 19 on all lymphocytes and on both CD4+ and CD8+ lymphocytes independently. Alteration of the V beta chains in HIV+ disease was analyzed as a function of absolute CD4 count and Centers for Disease Control (CDC) stage of the patient. These data suggest that the loss of T helper (CD4) lymphocytes during the course of HIV disease may be a selective event. These data are consistent with the hypothesis that selective depletion of CD4+, V beta 19+ lymphocytes may be due to the encoding of a superantigen by HIV. Furthermore, using multicolor flow cytometry and stratifying patients by absolute CD4 counts (or stage of disease) may reveal immunologic changes that might otherwise be overlooked.

Adult↗
PubMed1995

Products of cells from gliomas: IX. Evidence that two fundamentally different mechanisms change extracellular matrix expression by gliomas.

Four human astrocytic gliomas of high grade of malignancy were each evaluated in tissue and in vitro for percentages of cells expressing glial fibrillary acidic protein (GFAP), collagen type IV, laminin and fibronectin assessed by immunofluorescence with counterstaining of nuclear DNA. Percentages of cells with reticulin and cells binding fluorescein-labeled Ulex europaeus agglutinin were also assessed. In tissue, each extracellular matrix (ECM) component was associated with cells in the walls of abnormal proliferations of glioma vessels, and all four tumors had the same staining pattern. Two strikingly different patterns of conversion of gene product expression emerged during in vitro cultivation. (1). In the most common pattern, percentages of all six markers consistently shifted toward the exact phenotype of mesenchymal cells in abnormal vascular proliferations: increased reticulin, collagen type IV, laminin and fibronectin; markedly decreased glial marker GFAP and absent endothelial marker Ulex europaeus agglutinin. The simplest explanation of this constellation of changes coordinated toward expression of vascular ECM markers is that primary glioma cell cultures are overgrown by mesenchymal cells from the abnormal vascular proliferations of the original glioma. These cell cultures were tested for in situ hybridization (ISH) signals of chromosomes 7 and 10. Cells from one glioma had diploid signals. Cells from the other glioma had aneuploid signals indicating they were neoplastic; however, their signals reflected different numerical chromosomal aberrations than those common to neoplastic glia. (2). The second pattern was different. Cells with ISH chromosomal signals of neoplastic glia retained GFAP, and gained collagen type IV. Their laminin and fibronectin diminished, but persisted among a lower percentage of cells. Cloning and double immunofluorescence confirmed the presence of individual cells with glial and mesenchymal markers. A cell expressing GFAP in addition to either fibronectin, reticulin or collagen type IV is not a known constituent of glioblastoma tissue. This provides evidence of a second mechanism of conversion of gene expression in gliomas.

Astrocytoma↗
PubMed1995

Flow cytometric analysis of primary and metastatic squamous cell carcinoma of the oral cavity and oropharynx.

A retrospective analysis of formalin-fixed, paraffin-embedded tissue from patients with histologically confirmed metastatic squamous cell carcinoma was performed using flow cytometry. Ninety-eight sets of specimens from previously untreated patients with an oral cavity or oropharyngeal tumor and a simultaneous cervical metastatic deposit were analyzed. Normal mucosa and cervical lymph nodes were processed identically and run as controls. All patients underwent surgical resection at Wilford Hall USAF Medical Center or The Eye and Ear Hospital of Pittsburgh between 1980 and 1986. The specimens from 94 patients were technically adequate for interpretation. Diploid histograms in both the primary and metastatic tumors were present in 49 (52%) of 94 patients. Aneuploid histograms in either the primary and metastatic tumors were noted in 45 (47%) of 94 patients. In this group of 45 patients, the primary tumor and cervical metastasis were both aneuploid in 21 (46%), and aneuploid histograms occurred with equal incidence in either the primary or metastasis in the remaining 24 cases. No statistically significant prediction of survival could be made from any correlation with the histograms of either the primary or metastasis. The potential technical problems and limitations of flow cytometry in the determination of DNA content of formalin-fixed, paraffin-embedded tissue and the selection of patients with advanced disease warrant caution in the interpretation of results.

Carcinoma, Squamous Cell↗
PubMed1995

Detection and monitoring of a concomitant atypical myeloproliferative disorder and chronic lymphocytic leukemia by flow-cytometric immunophenotyping.

OBJECTIVE: To illustrate the utility of a broad panel of monoclonal antibodies to detect secondary processes or unexpected characteristics of the primary blood dyscrasia. DESIGN: Case report and discussion. SETTING: Regional academic medical center. PATIENT: A 64-year-old man presenting with an apparent acute myeloid leukemia. INTERVENTIONS: Sequential immunophenotyping with a broad panel of monoclonal antibodies to monitor progression of disease and response to therapy. MAIN OUTCOME MEASURE: Identification and monitoring of the two atypical populations in this patient with correlation to the clinical status of the patient. RESULTS: Identification of an unsuspected mature lymphoid clone and characterization of the evolution of the myelomonocytic clone. CONCLUSION: The evolving mature lymphoid clone may have been overlooked in the context of a predominant atypical myeloproliferative process, particularly if a limited panel of monoclonal antibodies had been used for immunophenotyping. Sequential immunophenotyping was useful in monitoring the progression of each atypical process.

Antigens, CD↗
PubMed1994

Quality control in flow cytometry for diagnostic pathology: II. A conspectus of reference ranges for lymphocyte immunophenotyping.

Immunophenotyping, as many other clinical assays, is interpreted only in the context of reference values obtained from healthy control individuals. While the use of these reference values, or ranges, has been commonplace in the clinical flow cytometry laboratory for well over a decade, there has been little consensus in standardizing how these values should be obtained, analyzed, or expressed. This report reviews the variables to be considered in establishing reference ranges and statistical methods which can be used. Additionally, examples are given of previously published reference ranges for a variety of specimens often submitted for immunophenotyping.

Adolescent↗
PubMed1994

Abnormal cytokine expression in Sézary and adult T-cell leukemia cells correlates with the functional diversity between these T-cell malignancies.

The Sézary syndrome (SzS) and adult T-cell leukemia (ATL) are malignant proliferations of mature T-lymphocytes that possess distinct functions. Sézary cells function as helper cells, whereas ATL cells are usually suppressor effectors. Although phenotypically similar (CD4+/CD7-/CD8-), these functional differences between the T-cell lymphoproliferative disorders suggest different patterns of cytokine expression. We wished to delineate the cytokine mechanisms potentially underlying the diverse functional characteristics of SzS and ATL. Therefore, we analyzed the expression of interleukins (IL) 2, 4, and 5, gamma-interferon, and transforming growth factor beta 1 in the highly purified leukemic T-cells from 5 SzS and 5 ATL patients. Decreased mRNA and protein levels of IL-2, gamma-interferon, and IL-5 were detected in mitogen-stimulated ATL and SzS cells when compared to similarly cultured normal CD4+ cells. In contrast, IL-4 production was markedly up-regulated in the leukemic cells of 4/5 SzS patients as compared to ATL and normal controls. Finally, fresh ATL cells secreted higher levels of transforming growth factor beta 1 into the culture medium than the malignant T-cells from SzS patients. Collectively these results suggest that, similar to the murine CD4-expressing T-cell subsets Th1 and Th2, different cytokine profiles exist in a human population of CD4+ T-cells. Moreover, the distinct patterns of IL-4 and transforming growth factor beta 1 expression by SzS and ATL cells, respectively, are most consistent with the functional differences (i.e., helper versus suppressor activity) between these T-cell malignancies and thus may play important roles in the pathogenesis of the paraneoplastic features associated with these two leukemias.

Adult↗
PubMed1994

Reversing the effect of formalin on the binding of propidium iodide to DNA.

Formalin, an excellent preservative of cellular morphology, is a commonly used fixative for tissue specimens in hospital pathology laboratories. This preserved material is a potential source of tissue for diagnostic and retrospective research studies on DNA using flow cytometry. Unfortunately, formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA, thus altering the measurement of DNA content by flow cytometry or image analysis. This interference has been attributed to the cross-linking of histones by formalin. Since formalin alters the measurement of DNA content in formalin-fixed and formalin-fixed, paraffin-embedded tissues, this study was designed to explore the use of various physicochemical methods to reverse the effect of the formalin on the binding of PI to DNA. This study demonstrates that resuspending formalin-fixed cells in PBS and heating them at 75 degrees C for at least 1 h prior to staining with PI restores the staining of the DNA to approximately the same fluorescence intensity as that of fresh tissue.

Artifacts↗
PubMed1994

Accuracy and cost-effectiveness of a one-tube, three-color method for obtaining absolute CD4 counts and CD4:CD8 ratios.

An increasing number of assays to determine absolute CD4 counts are being performed as the acquired immunodeficiency syndrome epidemic continues. Simultaneously, there is increasing pressure to contain costs in the clinical laboratory. A rapid, one-tube, three-color method for obtaining CD4 counts has been evaluated for accuracy, compared with a more traditional two-color panel of markers. The data, compiled on 102 patients, including both pediatric and adult human immunodeficiency virus-infected patients, indicate a high degree of correlation between the one-tube method and a traditional panel. This approach should be considered for cost-effective and accurate CD4 determinations.

Adult↗
PubMed1994

Flow cytometric analysis of growth hormone-secreting pituitary adenomas.

Flow cytometric DNA analysis was performed on samples of paraffin-embedded and fresh adenomatous pituitary tissue removed from 16 patients with growth hormone (GH)-secreting pituitary adenomas. Histograms consistent with aneuploidy were found in 5 of 16 patients. The flow cytometric patterns on fresh and paraffin-embedded tissues yielded similar results. Ploidy did not correlate with the clinical course or the endocrine profiles in these patients. We conclude that flow cytometric DNA analysis can detect aneuploidy in adenomatous pituitary tissue, but it does not predict the clinical course of GH-secreting adenomas.

Adenoma↗
PubMed1993

Isolation and purification of a squamous cell carcinoma of the head and neck-associated antigen identified by autologous antibody.

We have previously shown that detection of autologous antibody activity to squamous cell carcinoma of the head and neck may be augmented by dissociation of immune complexes. Western blot analysis with autologous antibody has identified a 60 kDa squamous cell carcinoma of the head and neck-associated antigen in spent media and immune complex-dissociated serum ultrafiltrate not recognized by normal human sera. Antigen-containing fractions of spent media were eluted from anion exchange columns immediately after serum albumin indicating that the antigen has an acidic pI < 4. Preparative purification of the squamous cell carcinoma antigen was accomplished by anion exchange of concentrated spent media (protein concentration 300 mg/ml) followed by lectin affinity chromatography with a Triticum vulgaris column. A single 60 kDa band was detected by silver stain and Western blot in antigen-containing fractions eluted following lectin affinity chromatography and SDS-PAGE. Final concentration of the antigen was determined to be 1 microgram/ml of protein with relative activity increased 1600 x over unfractionated spent media. We conclude that a squamous cell carcinoma of the head and neck-associated antigen, detected by autologous antibody, is an acidic 60 kDa glycoprotein.

Antibodies↗