George B. Olson

Correlation between allergy and persistent Epstein-Barr virus infections in chronic-active Epstein-Barr virus-infected patients.

Forty-six anti-Epstein Barr nuclear antigen-positive allergic patients, 11 of whom having clinical and laboratory evidence of chronic-active Epstein-Barr virus (CA-EBV) infections, were characterized by EBV serology, percentages of T cells, B cells, and IgE+ cells, serum levels of IgE, and allergen-induced responsiveness of lymphocytes. Results demonstrated patients with CA-EBV have significantly increased responsiveness toward specific allergens, responses toward greater numbers of allergens, numbers of IgE+ T and B cells, and levels of background DNA activity in nonstimulated lymphocytes than do subjects who suffer from allergies in the absence of the CA-EBV syndrome. Further comparison between subjects with laboratory-determined mild and moderate allergy and those with CA-EBV demonstrated a progressive increase in the serum levels of IgE as the degree of allergy increased, no difference in concentrations of T and B cells, and titers of anti-viral capsid antigen and anti-early antigen to be significantly greater in patients with CA-EBV. Statistical analysis demonstrated that patients with CA-EBV could be separated from subjects with allergies by metabolic and immunologic variables. The data suggested that allergen-induced responses may contribute to the CA-EBV syndrome.

Computer Analysis of Lymphocyte Images

Research on computer analysis of microscopic images of cells concentrates at this time on three major applications. These are the automated recognition of cells from the hematopoietic system, the analysis of epithelial cells and the study of lymphocyte populations. Of these, the first two are clearly related to the immediate needs of the clinical laboratory. White blood cell differential counts are carried out at a rate of more than 100 million per annum in the United States alone. Research and development here has progressed from the first feasibility studies accomplished in the 1960s by Preston (1961) and Preston (1962), Prewitt and Mendelsohn (1966), Ingram et al. (1968), Young (1969), and Bacus (1970) to the commercially available automated white blood cell differential counting devices. For the analysis of epithelial cells, the major effort has concentrated on the clinical cytology of the female reproductive tract (Wied et al., 1976). Here, research toward the improvement of the diagnostic characterization of the material and extensive efforts to automate the prescreening for cervical cancer are underway. Also directed toward early detection and diagnosis of malignant disease, and particularly toward the extraction of prognostic clues, are research projects on image analysis of urothelial cells (Koss et al., 1975, 1977a,b, 1978a; Bartels et al., 1977c) and cells from the respiratory tract (Reale et al., 1978; Wied et al., 1979). The analysis of digitized images of lymphocytes at this time does not have such immediate clinical applications.

EVALUATION OF CORRELATIONAL INFORMATION IN DIGITIZED CELL IMAGES

The processing of digitized cell images by computer may yield information not only about a set of cell image properties but also about their mutual dependencies. Observation of the covariance matrix of image properties of cellular material showing a response to chemotherapeutic treatment, ionizing radiation or antigenic challenge or following a developmental trend may permit a quantitative description of small trendal charges The covariance between certain cell image properties may show statistically significant changes before the mean values of the image properties are affected. Methods of reducing the dimensionality of the representation in an efficient manner are described.

Computer analysis of defined populations of lymphocytes irradiated in vitro: III. Evaluation of human T and B cells of peripheral blood origin*

The radiosensitivities of lymphocytes of peripheral blood origin obtained from two healthy 45 year old male donors were studied simultaneously at yearly intervals over a three year period. Hypaque-Ficoll purified cells were exposed in vitro to 0, 5, 50, and 500 rads and then evaluated serially for viability of T and B cells, responsiveness to PHA and Con A, and morphologic evidence of injury as documented by standard light microscopy and computer assisted morphometric analysis. The results showed that T cells in both subjects were less radiosensitive than B cells. Differences between the two subjects also existed in the radiosensitivity of these two subpopulations of lymphocytes, differences that remained constant over the three years period of observation. The differences correlated with similar discrepancies in mitogenic responsiveness and are thought to relate to variations in the relative proportions of subpopulations of T and B cells. In the mouse, T and B cell subpopulations differ in radiosensitivity. The data reported herein are consistent with a similar situation in man.

Differentiation of murine thoracic duct lymphocytes into t and b subpopulations by computer cell-scanning techniques.

Abstract Computer-assisted cytometric analysis of murine lymphocytes, and of pure populations of T and B cells obtained from thoracic duct lymphocytes (TDL) revealed the existence of descriptors capable of differentiating T and B lymphocytes. Optical density values of Feulgen-stained nuclear DNA of the cells were recorded as digitized images by a computerized scanning microscope. Computer programs extracted from each cell and for each cell population computable image information related to the DNA distribution patterns. Nuclei of B cells possess smaller areas, less total optical density, denser staining DNA granules than the nuclei of T cells and distinct differences in the arrangement of optical density values. These descriptors allowed the differentiation of TDL into two subpopulations possessing properties identical to those found in pure T and B cell populations.

Specific allergen-induced Epstein-Barr nuclear antigen-positive B cells from patients with chronic-active Epstein-Barr virus infections

Enriched B cells and peripheral blood lymphocytes from patients with chronic-active Epstein-Barr virus (CA-EBV) infections and subjects with mild and moderate allergies were cultured in vitro with specific allergens known to cause allergic reactions. A significant increase in Epstein-Barr nuclear antigen+ cells occurred only in the B cells obtained from patients with CA-EBV when cells were stimulated with the specific antigen. Results indicate an association between EBV-transformed cells and B cells with idiotypic expressions and may help to explain the association between CA-EBV and allergy in these patients.

Cytophotometric Studies of Cell Populations

Technologic advances in the recording of digitized imagery have made the study of large cell populations by image analytical methods feasible. Computed image information provides quantitative, and novel, information that allows an exact measurement of minute changes in the chromatin distribution of cell nuclei, and the detection of subpopulations of cells or changes in the functional state of the cells. The sensitivity of the detection exceeds that of human observers; the specificity of the measured changes must be the subject of basic cell biologic research.

The automated analytical electrophoresis microscope

The components of an automated computer-controlled analytical electrophoresis microscope (AEMS) are described. Computer tracking of migrating cells projected under phase contrast onto a vidicon permits the rapid taking of multiple velocity measurements per cell so that reliable determinations of electrophoretic mobilities thereby result. The computer-controlled cell search and tracking algorithms allow high speed operation so that statistically valid profiles and data bases can be collected rapidly. Initial electrophoretic mobility evaluations have been carried out on populations of lymphocytes, erythrocytes, and platelets.

Characterization of murine T and B cells by computerized microphotometric analysis

Splenocytes and column-separated T cells are differentiated into subpopulations of T and B cells on the basis of computer-assisted morphometric analysis of Feulgen-positive nuclear DNA. Differentiation is based upon the analysis of computable image information related to DNA distribution patterns. The technique at the present time does not allow immunofluorescent and morphometric measurements to be made on a given cell. However, the differentiation obtained by using descriptors proven capable of detecting pure populations of T and B cells shows excellent agreement with the differentiation obtained by immunofluorescence analysis. The descriptors and decision rules used in the discrimination among splenocytes are reproducible from one experiment to another and remain valid for the differentiation of lymphocytes from animals of different sex and strain.

Viral-Induced Unresponsiveness of Tuberculin-Sensitized Guinea Pig Lymphocytes

Summary Blast cell transformation in vitro of lymphocytes obtained from the peripheral blood of guinea pigs after sensitization to mycobacterial antigen was shown to increase after PPD stimulation. However, the cellular activity of lymphocytes was influenced by recent sensitization or immunization of the animals. The blastogenic response of guinea pig lymphocytes to the mitogen PHA was decreased following both the initial tuberculin sensitization and skin testing with PPD. Lymphocytes from tuberculin-sensitized animals demonstrated a fall in responsiveness to in vitro PPD stimulation after the animals were skin tested. Furthermore, this study shows that in vitro NDV infection of sensitized lymphocytes prevents the cells from undergoing PPD-induced transformation. This confirms earlier work on viral-induced lymphocyte unresponsiveness and establishes the guinea pig as an animal model for investigating the virus-lymphocyte relationship.