Robbin DeBiasio

High-Content Screening: A New Approach to Easing Key Bottlenecks in the Drug Discovery Process

Recent improvements in target discovery and high throughput screening (HTS) have increased the pressure at key points along the drug discovery pipeline. High-content screening (HCS) was developed to ease bottlenecks that have formed at target validation and lead optimization points in the pipeline. HCS defines the role of targets in cell functions by combining fluorescence-based reagents with the ArrayScan™ System to automatically extract temporal and spatial information about target activities within cells. The ArrayScan System is a tabletop instrument that includes optics for subcellular resolution of fluorescence signals from many cells in a field within a well of a microtiter plate. One demonstrated application is a high-content screen designed to measure the drug-induced transport of a green fluorescent protein-human glucocorticoid receptor chimeric protein from the cytoplasm to the nucleus of human tumor cells. A high-content screen is also described for the multiparametric measurement of apoptosis. This single screen provides measurements of nuclear size and shape changes, nuclear DNA content, mitochondrial potential, and actin-cytoskeletal rearrangements during drug-induced programmed cell death. The next generation HCS system is a miniaturized screening platform, the CellChip™ System, that will increase the throughput of HCS, while integrating HCS with HTS on the same platform.

Quantification of G-Protein Coupled Receptor Internalization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScan™ High-Content Screening System:

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScan™ (Cellomics™, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathy-roid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a β2-adrenergic receptor (β2 AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate spots within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.

Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD).

Progress has been made in improving the immunohistochemical detection of antigens for imaging and flow cytometry. We report the synthesis of a novel fluorescent horseradish peroxidase substrate, Cy3.29-tyramide, and its application in an enzyme-based signal amplification system, catalyzed reporter deposition (CARD). The catalyzed deposition of Cy3.29-tyramide was used to detect cell surface markers such as CD8 and CD25 on tonsil tissue and human lymphocytes. We compared the fluorescence CARD method to standard indirect immunofluorescence detection methods and found that an amplification of up to 15-fold was possible with CARD. The detection of the intracellular protein myosin II in fibroblastic cells and rabbit serum proteins blotted onto nitrocellulose was also improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance significantly the detection of antigens.

Potential of Machine-Vision Light Microscopy in Toxicologic Pathology:

Major developments in machine-vision light microscopy and in reagent chemistry have led to a renaissance and revolution in the use of the light microscope in biology, biotechnology, and medicine. The potential use of this technology in the field of toxicologic pathology is discussed. It is suggested that a combination of investigating living cells and tissues and fixed samples using the new technologies will lead to understanding mechanisms of toxicity. Examples of the use of the methods in basic cell biology and medicine are presented.

Alteration of immunoregulatory mechanisms during cytomegalovirus mononucleosis: Effect of in vitro culture on lymphocyte blastogenesis to viral antigens

Immunoregulation of lymphocyte blastogenesis was studied in 13 patients with acute-phase cytomegalovirus (CMV) mononucleosis and 9 of these patients during the convalescent phase of the illness. Peripheral blood mononuclear leukocytes from acute-phase patients displayed depressed uptake of [3H-]thymidine in response to the lectin-mitogen concanavalin A (Con A) and immune-specific viral antigens (CMV, herpes simplex virus (HSV), mumps virus) compared with convalescent patients or normal donors. Removal of plastic-adherent cells from the patients samples resulted in further depression of lymphocyte blastogenesis to Con A and CMV and HSV antigens, suggesting a helper function for the predominantly monocytic, adherent cell population in this response. Preliminary culture of mononuclear leukocytes from acute-phase patients for 18 hr at 37 degrees C resulted in significantly enhanced blastogenesis to Con A. In sharp contrast, lymphocyte blastogenesis to viral antigens was not significantly enhanced after preculture. These results suggest that different mechanisms are operative in immunoregulation of lymphocyte recognition responses to the polyclonal activator Con A and immune-specific viral antigens during human CMV infection.

Automated interactive microscopy: measuring and manipulating the chemical and molecular dynamics of cells and tissues

The Automated Interactive Microscope is a robotic light microscope workstation that combines high performance light microscopy and computing to explore the chemical and molecular dynamics of cells and tissues.

Automated Light Microscopy for the Study of the Brain: Cellular and Molecular Dynamics, Development, and Tumorigenesisa

The impressive advances in technology within the last few decades have had a major impact on the field of microscopy. This is particularly true of biological light microscopy, where research has benefited from a convergence of developments in fields as diverse as electronics, optics, molecular biology, computer science, robotics. and reagent chemistry.. The integration of these developments has provided a dynamic research tool that puts within reach the elucidation of key mechanisms of biological functions, by noninvasive, high-resolution. quantitative monitoring of living cells, tissues, and organisms.j By adding optical and electrical micromanipulation techniques, one can simultaneously measure and modify cell One can hardly think of a research field for which such possibilities are more exciting than the study of the brain, where these tools now permit analysis from the subcellular to the fully functional level. Research initiated about a century ago by Santiago Ram6n y Cajal and his contemporaries showed that the nervous system is made up of individual cells interconnected and functioning by virtue of the structure of the neural networks.6 He was able to make these astounding interpretations by capitalizing on new techniques for staining tissue developed in his day by Camillo Golgi and new technology invented by Carl Zeiss, that improved the light microscope. Today, another renaissance in our understanding of the nervous system is occurring, again due to new methods for staining and new technologies for optical imaging and visualization of events as they occur in cells and tissues. We can now peer into

Effect of herpesvirus infections on T-lymphocyte subpopulations and blastogenic responses in renal transplant recipients receiving cyclosporine

The immunosuppressive effects of three herpesviruses--cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV)--were assessed in 29 renal transplant recipients treated with cyclosporine and prednisone. The ratios of Leu 3-positive (helper-inducer) to Leu 2-positive (suppressor-cytotoxic) T lymphocytes in peripheral blood were only moderately and transiently decreased during primary CMV infection, with or without concurrent reactivated EBV and HSV infections. This effect was due to an increase in absolute numbers of Leu 2-phenotypic and decrease in Leu 3-phenotypic T cells and was associated with symptomatic viral illness. Reactivated CMV infection alone or together with reactivated EBV and HSV infections resulted in less significant alterations in T-cell subsets than did primary CMV infection. Lymphocyte blastogenesis was not significantly altered during the herpesvirus infections. The data suggest that cyclosporine treatment inhibits the activation of suppressor cells and depression of cellular immune function that have been associated with herpesvirus infections in renal transplant recipients undergoing conventional immunotherapy.

VIROLOGIC, IMMUNOLOGIC, AND EPIDEMIOLOGIC ASSOCIATIONS WITH AIDS AMONG GAY MALES IN A LOW INCIDENCE AREA a

The Pittsburgh tri-state area is presently a low incidence area for AIDS and AIDSrelated complex (ARC). There are only six confirmed cases of full-blown AIDS using the Centers for Disease Control criteria. The first case occurred in 1980, the second appeared in 1982, and four were discovered in 1983, which is two cases per million in the Pittsburgh area. This is lower than the 5 to over 30 cases per million noted in such cities as Los Angeles, San Francisco, Miami, Houston, and New York. We estimate that there are only about 20 known cases of ARC in Pittsburgh. We have confirmed 10 of these in our study using the definition of noncontiguous, extrainguinal lymphadenopathy persistent for more than three months, without any association with known, non-AIDS-related causes. All cases of AIDS and ARC have been in non-Haitian gay males with no history of intravenous drug abuse. We began our pilot study earlier this year to examine the basis for this low incidence of AIDS. There are two major hypotheses that could explain the low incidence. First, the putative infectious agent or agents causing AIDS and ARC may not be widely introduced into our gay population. Second, and not necessarily exclusive from the first, is that various lifestyle, virologic, and immunologic characteristics that have been associated with AIDS differ between gay males in Pittsburgh and those in high incidence areas. Our study population consisted of 57 gay male volunteers who were healthy by physical exam and history. These subjects were all white and were predominantly in their 20s and 30s. The healthy subjects began having gay sex in their late teens. They were relatively sexually active as evidenced by an average of over 30 different sex partners in the year preceding our study. About one-third of our subjects participated in sex at gay baths. Those studied were also sexually active as shown by a relatively high level of self-reported episodes of sexually transmitted diseases (mean of 3) and history of hepatitis (26%). Over 60% participated as the passive recipient in anal intercourse.

Approaches to the Study of Spatial and Temporal Changes in the Structure and Chemistry of Cells

Publisher Summary This chapter presents the study of spatial and temporal changes in the structure and chemistry of cells. The chapter also presents requirements of multiple spectral parameter imaging. It is evident that most cellular functions involve the temporal and spatial integration of changes in ionic parameters, metabolites, macromolecules, and organelles. Therefore, a method has been developed to correlate the temporal and spatial variations in up to five separate parameters based on the spectral isolation of distinct fluorescent probes. This method allows the integration of results from distinct measurements of parameters such as pH, free calcium concentration, membrane potential, specific fluorescent analogs, and whole organelles. The chapter explains the structural and chemical changes in quiescent Swiss 3T3 cells during the polarized spreading and the movement following activation by creating a wound.