Philip F. Mixter

Phosphorylation of the cell cycle inhibitor p21Cip1/WAF1 by Pim-1 kinase.

The serine/threonine kinase, Pim-1, appears to be involved in regulating proliferation, differentiation and cell survival of lymphoid and myeloid cells. In this study, we have found that amino acid residues 140-147 (RKRRQTSM) at the C-terminal end of p21(Cip1/WAF1), a cyclin-dependent kinase (CDK) inhibitor, constitute an ideal phosphorylation consensus sequence for Pim-1. We demonstrate that Pim-1 efficiently phosphorylates this peptide sequence as well as the p21 protein in vitro. We also demonstrate by pull-down assay and by immunoprecipitation that Pim-1 associates with p21. During phorbol ester-induced differentiation of U937 cells, both Pim-1 and p21 expression levels increase with Pim-1 levels increasing in both the nucleus and cytoplasm while p21 remains primarily cytoplasmic. Co-transfection of wild type p21 with wild type Pim-1 results in cytoplasmic localization of p21 while co-transfection of wild type p21 with kinase dead Pim-1 results in nuclear localization of p21. Consistent with the results from the phosphoamino acid assay, Pim-1 phosphorylates transfected p21 only on Thr(145) in p21-deficient human fibroblasts and this phosphorylation event results in the cytoplasmic localization of p21. These findings demonstrate that Pim-1 associates with and phosphorylates p21 in vivo, which influences the subcellular localization of p21.

The origin of CD4−CD8−TCRαβ+ thymocytes: a model based on T-cell receptor avidity

Ralph Budd and Philip Mixter present a hypothesis suggesting that CD4-CD8-TCR alpha beta+ cells arising in the normal thymus result from a high-avidity T-cell receptor (TCR) signal bordering on negative selection. In normal mice, this subset is likely to be gradually deleted but, in the absence of Fas, these cells persist in lpr mice. The model they describe makes several predictions regarding the nature of this unusual T-cell subset.

In Vivo Tracking of Campylobacter jejuni by Using a Novel Recombinant Expressing Green Fluorescent Protein

ABSTRACT Campylobacter jejuni is a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study of C. jejuni in a variety of niches. C. jejuni transformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally with C. jejuni harboring the gfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined that C. jejuni synthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b+ Gr-1+ lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b+ Gr-1− lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b− CD45R+ B lymphocytes from the lavage of the C. jejuni-injected mice were associated with green-fluorescent C. jejuni (proportions ranged from 0.75 to 0.77%). Cell-free C. jejuni was recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of the C. jejuni F38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary, we demonstrate that C. jejuni associates with marker-defined cellular subsets in vivo with a novel gfp reporter system and that C. jejuni genotypic variants can be isolated from both in vitro and in vivo systems.

Flow cytometric detection of host cell apoptosis induced by bacterial infection

We detail two methods for detection of cell death induced by infection of a human monocytic cell line with invasive Campylobacter bacteria. Staining with a natural ligand for exposed phosphatidylserine residues coupled with propidum iodide discriminated between apoptosis and necrosis. Additionally, cells infected with a bacterial strain expressing green fluorescent protein stained with dye sensitive to mitochondrial membrane potential demonstrated a direct association of bacteria with dying cells. Analyses of cells stained by these methods employing flow cytometry enumerated proportions of cell populations undergoing either apoptosis or necrosis after bacterial infection in vitro.

Role-Playing in a Vaccination Debate Strengthens Student Scientific Debate Skills for Various Audiences †

Students are surrounded by strongly-held viewpoints on scientific topics and frequently discuss news reports with their classmates. We developed the vaccination debate exercise to leverage this interest and develop core higher-order cognitive skills (HOCS), including, but not limited to, the ability to critique public media or primary research sources and create arguments for defending multiple viewpoints. Students prepared to debate different sides of the topic and then randomly assumed one of the roles: “Physician” (pro-vaccine), “Activist” (anti-vaccination), or “Parent-on-the-fence” (undecided). Students reported an increase in their abilities to discuss scientific topics with diverse audiences and an increased awareness of importance of examining Internet sources for credibility.

Fundamental Immunology (4th edn) edited by W.E. Paul

Lippincott-Raven Publishers, 1999.

Decreased CD4-CD8- TCR-alpha beta + cells in lpr/lpr mice lacking beta 2-microglobulin.

135.00 (xi + 1589 pages)ISBN 0 7817 1412 5In assembling the fourth edition of Fundamental Immunology, W.E. Paul strives to cover the collective landscape of the field. This edition has been reorganized first to introduce concepts and review history, before moving on to chapters detailing the current status and future trends in focused areas. The reorganization is designed to help readers with a limited background in the field gain insight, but few should use this book as an introductory text. However, for readers with some background or with main interests in related areas, and/or those desiring a current reintroduction to the field, this fourth edition will supersede Pauls goal of providing an ‘authoritative presentation of the basic elements and mechanisms of clinical conditions’.Fundamental Immunology represents a superb collection of chapters pertinent to any individual interested in gaining current information about immunology. The 45 chapters are written by leading researchers in their respective areas of expertise. These chapters build from fundamental cell biology, tissue organization and immunoregulation to an integrated view of the mechanisms underlying clinical immunology. The individual chapters vary slightly in their organization and focus, but most include a comprehensive review of current thinking in the specific area, with some indication of the outstanding questions.Many readers may use a volume of this size and scope for reference. With this purpose in mind, the organizational annotation is limited to a table of contents and a brief chronological outline at the beginning of each chapter. Additional notation of page numbers for subsections within the chapter would benefit readers trying to find specific, detailed information. Alternatively, an enhanced and expanded index with more precise terms could eliminate this deficiency.The fourth edition of Fundamental Immunology is a great resource covering a broad range of topics in depth. The individual contributors bring their own insight and bias with the benefits of their expertise. While some may choose to dwell on the primary literature for the most current information, those looking to gain a comprehensive and current view of immunology in one volume will find this book a most practical purchase.