Philip J. Morrissey

Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer.

Here we demonstrate the ability of the ImageStream® 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features.

Sensitivity measurement and compensation in spectral imaging

The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented.

Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.

Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein.

ABSTRACT The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.

Development of a recombinant growth factor and fusion protein: lessons from GM-CSF.

Several colony stimulating factors (CSFs) and cytokines have been successfully used to mobilize hematopoietic cells during myeloablative therapy, bone marrow failure, and transplantation and to provide supportive treatment during sepsis. The use of yeast-derived recombinant human granulocyte-macrophage CSF (rhuGM-CSF) and its interleukin-3 fusion protein, PIXY321, provides an example of issues associated with development programs for recombinant hematopoietic growth factors. Species specificity of rhuGM-CSF, different bioactivity of homologous molecules in mice, and production in laboratory animals of antibodies to human proteins limit preclinical evaluation of such molecules. In clinical trials, rhuGM-CSF was efficacious and well tolerated. The derivation of the recombinant molecule, optimal dosing, scheduling, and confounding effects of concurrent disease and treatments are factors that influence efficacy, adverse responses, and immunogenicity reported in patients treated with CSFs. In comparisons of yeast-derived with Escherichia coli-derived rhuGM-CSF, the reduced severity and frequency of all adverse events, preponderance of low-grade adverse events, and similarity of positive clinical response versus adverse events reported for granulocyte CSF support safety and efficacy of yeast-derived rhuGM-CSF Enhanced pharmacoeconomic evaluations are beginning to limit and redirect clinical applications in this class of biological agents.

Lymphoid Development and Function in IL-7R-Deficient Mice

The stages of B- and T-cell progenitor development have been thoroughly defined on the basis of cell surface marker expression, antigen receptor gene rearrangement, and cytokine responsiveness. For example, interleukin-7 (IL-7) was originally identified as a factor produced by bone marrow stromal cells that could support the proliferation of B-cell precursors (1,2). The IL-7 responsive B-lineage cells were clearly defined in vitro using sorted B-cell precursor populations and shown to express B220, CD43 heat stable antigen (HSA), and to carry heavy-chain gene rearrangements (3). IL-7 responsiveness is lost following light-chain gene rearrangement and acquisition of the pre-B-cell phenotype (4). IL-7 is also a potent mitogen for thymocytes (5). In a similar fashion, it has been established that the CD44+CD25− fraction of CD4−CD8− thymocytes, comprising <1% of the total thymocyte population, is IL-7 responsive, whereas the predominant CD4+CD8+ thymocyte subpopulation is not (6).

Measurement of Cytoplasmic to Nuclear Translocation

A method is described for the quantitative assessment of the translocation of signaling molecules from the cytoplasm to the nucleus in cells. This method utilizes fluorochrome-conjugated antibodies to the signaling molecule and a nuclear dye, and it is based on imagery acquired rapidly in flow with the use of a multispectral imaging cytometer. The analysis correlates the spatial distribution of the stained translocating signaling molecule with nuclear staining, and it generates a quantitative score for each cell using Pearsons correlation coefficient. Examples described in this section use reagents that detect NFkappaB and IRF-7 and measure the translocation of these molecules under stimulating conditions. A protocol for combining cell surface phenotype with cytoplasm to nuclear translocation is also included.

Functional Phenotype of Mice Deficient in the IL-1R Type I Gene

Interleukin 1 (IL-1) is a cytokine that is involved in the mechanism of host resistance and immune function (for review, see ref. 1). Evidence indicates that IL-1 functions as a proinflammatory cytokine with significant and well-documented effects on innate resistance and the inflammatory response (2). The biological effects of IL-1 also influence the development of the specific immune response. IL-1 has been long studied as a costimulator of both B-cells and T-cells and the adjuvant effect of IL-1 is well documented (3–5). Thus, it is believed that IL-1 plays a central role in both innate resistance mechanisms and specific immune responses.