Gregory Beck

Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule.

That the plasma concentration of certain divalent cations change during an inflammatory insult provides a major host defense response in vertebrate animals. This study was designed to investigate the involvement of iron sequestration in invertebrate immune responses. A ferritin molecule was cloned from an echinoderm coelomocyte cDNA library. The amino acid sequence showed sequence homology with vertebrate ferritin. The cDNA contained a conserved iron responsive element sequence. Studies showed that stimulated coelomocytes released iron into in vitro culture supernatants. The amount of iron in the supernatants decreased over time when the amebocytes were stimulated with LPS or PMA. Coelomocytes increased expression of ferritin mRNA after stimulation. In vertebrates, cytokines can cause changes in iron levels in macrophages. Similarly, echinoderm macrokines produced decreases in iron levels in coelomocyte supernatant fluids. These results suggest that echinoderm ferritin is an acute phase protein and suggest that sequestration of iron is an ancient host defense response in animals.

Characterization of interleukin-1 activity in tunicates☆

1. Eight North American species of tunicates were examined for the presence of interleukin-1 (IL-1) like activity. 2. The tunicates studied produce molecules with readily detectable lymphocyte activation factor (LAF) activity. 3. G50 column chromatography separated molecular species that were directly mitogenic for thymocytes (mol. wt greater than 50,000) from those that were comitogenic in an IL-1 assay (mol. wt 20,000). 4. Tunicate fractions with LAF activity induced increased vascular permeability in rabbit skin. 5. Tunicate LAF activity was neutralized by polyclonal anti-human IL-1 antisera. 6. These data further support the conclusion that IL-1 is an ancient and functionally conserved molecule.

Primitive cytokines: harbingers of vertebrate defense

The evolution of the immune system has awarded cytokines a key role as coordinators of the immune response. The exquisite action of cytokines in fine tuning and controlling the response raises the question of whether or not these molecules have been highly conserved through evolution. Gregory Beck and Gail Habicht have isolated and characterized two major cytokines, interleukin 1 and tumor necrosis factor, from invertebrates. In this article, they speculate on the possible function of these molecules and on the existence of other cytokines in invertebrates.

Phylogenetic perspectives on the vertebrate immune system

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Isolation, preliminary chemical characterization, and biological activity of Borrelia burgdorferi peptidoglycan☆

Peptidoglycan (PG), an essential cell wall polymer of most bacteria, has been isolated from many species of spirochetes. Our interest in the host response to Borrelia burgdorferi led us to isolate and characterize its PG. Extracted cells were solubilized with warm 1% SDS followed by digestion with proteases. Amino acid analysis of the isolated PG demonstrated the presence of alanine, glycine, glutamic acid, and ornithine as occurs in other spirochetes and bacteria. Intense erythematous reactions were observed after id injection of 10 micrograms of PG into normal human skin. PG was not mitogenic for human peripheral blood mononuclear cells. Murine splenocytes of certain strains responded to the PG, but only at concentrations of 25 micrograms/ml or more. PG stimulated macrophages to produce interleukin 1. Sixteen micrograms of PG injected iv into rabbits produced biphasic fevers. These observations on the in vitro and in vivo activities associated with the cellular components of the B. burgdorferi spirochete give further insight to how a small number of invading organisms can cause a multisystemic disease such as Lyme disease.

A role for interleukin-1 in the pathogenesis of Lyme disease.

Summary Interleukin-1 (IL-1) is the major immunoregulatory molecule produced by macrophages in response to a variety of environmental insults including chemicals, phagocytosis, bacteria, and bacterial products. Macrophages stimulated by Borrelia burgdorferi produced large quantities of IL-1 when spirochetes were added to macrophages at a ratio of 10 spirochetes per macrophage. The release of IL-1 was dose dependent: a single spirochete per macrophage was sufficient to produce significant quantities of IL-1. Spirochetal lipopolysaccharide was not required for this activity in that polymyxin B in the spirochete-macrophage culture had no effect on IL-1 production. Normal murine fibroblasts cultured with this IL-1 were shown to have an increased rate of DNA synthesis and an increase in secreted collagenase. IL-1 was found in joint fluids from Lyme disease patients. When IL-1 was injected intradermally into the backs of rabbits, the injection sites became indurated, erythematous, and warm to the touch after 4 hrs and annular lesions much like those of erythema chronicum migrans were seen in some animals after 24 hrs. B. burgdorferi is a powerful inducer for IL-1 in vitro , and it is reasonable to presume that it acts similarly in Lyme disease patients. Our results suggest that IL-1 in turn, may play a role in many of the clinical manifestations of Lyme disease.

Isolation of the outer envelope from Borrelia burgdorferi

Borrelia burgdorferi consists of an inner protoplasmic cylinder, containing the genome and cytoplasmic elements, surrounded by a number of axial filaments, all completely encased within a multiple-layered outer envelope structure (OE). In this study, a sodium dodecyl sulfate-mediated technique was used to isolate the OE from Borrelia burgdorferi in an attempt to better understand this structure in terms of its antigenic reactivity to Lyme disease patient sera. Electron microscopic evidence suggested that the OE product was relatively free of other spirochete cellular components. SDS-PAGE analysis indicated that the electrophoretic pattern of the OE was consistent with that of the remaining protoplasmic cylinder (PC) and whole spirochete controls. Antigenic determinants in the OE were recognized by sera from Lyme disease patients in Western blots. Chemical analysis of the OE revealed a composition of 45.90% protein, 50.75% lipid and 3.33% carbohydrate. The OE comprised 16.5% by lyophilized dry weight of the whole spirochete. Antigenic determinants located within or associated with this structure are likely to play a significant role in the development of immunity in the infected host.

Mechanism of BCG-Activated Macrophage-Induced Tumor Cell Cytotoxicity: Evidence for Both Oxygen-Dependent and Independent Mechanisms

Activated peritoneal macrophages have been shown to be cytotoxic to cancer cells. Bacillus Calmette-Guerin (BCG)-activated rat peritoneal macrophages have a basal cytolytic potential for 3H-thymidine-labelled Walker 256 cancer cells in vitro that can be markedly enhanced by digitonin. This stimulation of cytotoxicity can be partially inhibited by catalase and the combination of superoxide dismutase plus catalase. This suggests that digitonin stimulates activated macrophages to produce superoxide, hydrogen peroxide and possibly other free radicals which can augment macrophage-induced tumor cell cytotoxicity. After a 2-hour incubation with digitonin, macrophages are no longer stimulated by digitonin. However, after a 2-hour drug preincubation period, inhibitors of serine protease activity (DFP, TLCK, SBTI) and inhibitors of protein synthesis (cycloheximide) are potent inhibitors of basal macrophage-induced tumor cytotoxicity. We suggest that BCG-activated macrophages have two mechanisms for destroying cancer cells: one mediated by proteolytic activity, and a second mechanism dependent on the generation of oxygen-derived free radicals.

Characterization of Tumor Cell Membrane Serine Proteases by Polyacrylamide Gel Electrophoresis

Studies in our laboratory have indicated that tumor cell membrane-bound proteases are responsible for the ability of tumor cells to lyse normal cells in vitro. In order to evaluate the tumor cell membrane enzymes, a purified tumor cell membrane preparation was prepared and the nonionic detergent Triton X-100 was used to extract active enzymes from the cell membranes. The solubilized membrane enzymes were then studied by Triton X-100 polyacrylamide gel electrophoresis under non-denaturing conditions. Using this technique the tumor cell membranes were shown to contain esterproteases that reacted with the substrates alpha-naphthyl acetate and naphthol-AS-aminocaproate. These esterproteases were inhibited by diisopropyl fluorphosphate and tosyl lysine chloromethyl ketone but not by tosylamide phenylethyl chloromethyl ketone, soybean trypsin inhibitor p-chloromercuribenzene sulfonic acid; N-ethylmaleimide choline iodide, alpha-1-anti-trypsin. NaF, epsilon-aminocaproic acid, ethylenediamine tetraacetic acid, or eserine. SBTI affinity chromatography of the tumor cell membrane extract revealed that some of the serine esterproteases bound to the SBTI column. The proteolytic activity of the tumor cell membrane extract and a fraction eluted from the SBTI affinity column was demonstrated using casein. We conclude that the tumor cell membranes contain previously undescribed serine proteases that are identifiable by their esterase activity and inhibitor profiles in polyacrylamide gels.