James F. Brown

Proteomic analysis of Schistosoma mansoni proteins released during in vitro miracidium-to-sporocyst transformation

Free-living miracidia of Schistosoma mansoni, upon penetration of the their snail intermediate host, undergo dramatic morphological and physiological changes as they transform to the parasitic sporocyst stage. During this transformation process, developing larvae release a diverse array of proteins, herein referred to as larval transformation proteins (LTPs), some of which are postulated to serve a parasite protective function. In the present study, nanoLC-tandem MS analysis was performed on all proteins represented in entire 1-dimensional SDS-PAGE-separated samples in order to gain a more comprehensive picture of the protein constituents associated with miracidium-to-sporocyst transformation and thus, their potential role in influencing establishment of intramolluscan infections. Of 127 proteins with sufficient peptide/sequence information, specific identifications were made for 99, while 28 represented unknown or hypothetical proteins. Nineteen percent of identified proteins possessed signal peptides constituting a cohort of classical secretory proteins, while 22% were identified as putative nonclassically secreted leaderless proteins based on SecretomeP analysis. Proteins comprising these groups consisted mainly of proteases/protease inhibitors, small HSPs, redox/antioxidant enzymes, ion-binding proteins including those with anti-oxidant Fe-binding activities (ferritins, heme-binding protein), and venom allergen-like (VAL) proteins. A polyclonal antibody generated against whole LTPs recognized proteins primarily associated with the cilia, ciliated epidermal plates and intercellular ridges of miracidia and the tegument of fully transformed sporocysts, identifying these structures as sources of a subset of LTPs. Thus lysis of plates and/or leakage during formation of the sporocyst syncytium likely represent significant contributors to the overall LTP makeup, especially identified nonsecretory proteins. However, as plate release/degradation and tegument formation are part of the normal developmental process, all LTPs regardless of tissue origin, would be expected at the parasite-host interface upon infection. This study significantly expands the repertoire of LTPs associated with larval transformation and identifies several, e.g., those involved in stress responses, proteolysis/inhibition, antioxidant and detoxication, and immune modulation, that may play a parasite protective role during this crucial period of transition.

Purified human and recombinant murine interleukin-1α induced accumulation of inflammatory peritoneal neutrophils and mononuclear phagocytes: possible contributions to antibacterial resistance

Interleukin-1 (IL-1) mediates a number of proinflammatory biological responses that are thought to contribute to antibacterial resistance. In the present study we examined the ability of IL-1 to recruit inflammatory neutrophils and mononuclear phagocytes in vivo; a function that has been reported to be closely related to antibacterial resistance. Intraperitoneal injection of small amounts (1-10 LAF units) of purified human or recombinant murine IL-1 alpha (rIL-alpha) resulted in an increased influx of inflammatory neutrophils into the peritoneal cavity that peaked at 4-14 h after IL-1 injection. A small but consistent increase in peritoneal macrophages also was observed at 72 h after rIL-1 alpha injection. The ability of rIL-1 alpha to induce neutrophil accumulation was uninfluenced by polymyxin B, was sensitive to heat treatment (100 degrees C for 1 h), and was observed after i.p. injection into LPS-nonresponsive C3H/HeJ mice. These three lines of evidence suggested that contaminating LPS did not contribute substantially to rIL-1 alpha induced accumulation of neutrophils. Treatment of mice with indomethacin or nordihydroguaiaretic acid did not abrogate rIL-1 alpha induced neutrophil accumulation. Mice injected i.p. with increasing amounts of rIL-1 alpha demonstrated a corresponding enhancement of their resistance to an i.p. L. monocytogenes challenge 4 h later, thus suggesting that IL-1 mediated inflammatory phagocyte accumulation may contribute in part to nonspecific antibacterial resistance.

Growth at reduced temperatures increases the virulence of Listeria monocytogenes for intravenously but not intragastrically inoculated mice

Growth of three clinical isolates (Scott A, Murray B, and F5380) and one laboratory strain (EGD) of L. monocytogenes at 4 degrees C significantly increased their virulence for intravenously injected mice. Using the EGD strain for subsequent experiments, we determined that growth at either 4 degrees or 22 degrees C enhanced the growth of listeria in the spleen and liver. Similar numbers of listeriae were recovered from the spleens and livers of mice during the first 48 h after i.v. injection of strain EGD grown at 37 degrees C or 4 degrees C. At later timepoints (3-6 days), significantly more listeriae were recovered from the spleens and livers of mice injected i.v. with strain EGD grown at 4 degrees C. In contrast, L. monocytogenes EGD grown at 37 degrees C and 4 degrees C demonstrated similar abilities to survive in the gastrointestinal tract, to translocate to the mesenteric lymph nodes, and to disseminate to the spleen and liver in intragastrically inoculated mice. Listeria monocytogenes EGD grown at 4 degrees C released less hemolysin into the culture medium than did this strain when grown at 22 degrees C and 37 degrees C. Transfer to fresh broth and incubation at 37 degrees C for 2 h increased the release, to similar levels, of hemolysin from L. monocytogenes EGD grown at 4 degrees, 22 degrees, and 37 degrees C. Temperature-induced differences in virulence, therefore, may not reflect the amount of hemolysin released.

Isolation and characterization of phenoloxidase from egg masses of the gastropod mollusc, Biomphalaria glabrata.

Previous studies have shown that phenoloxidase activity is present in the albumen gland and egg masses of Biomphalaria glabrata, and its potential role in egg formation in this snail has been proposed. In the present study, a phenoloxidase enzyme has been isolated from the supernatant of egg mass homogenates using a combination of hydrophobic interaction chromatography and gel filtration high-performance liquid chromatography (GF-HPLC). The isolated phenoloxidase eluted as a single peak of activity upon GF-HPLC (representing a 132-fold purification) and subsequently was detected as a single band with an estimated molecular mass of 35 kDa by SDS-PAGE analysis. Phenylthiourea-inhibitable mono- and diphenoloxidase activities were demonstrated for the isolated enzyme suggesting that both enzyme activities are associated with a single, tyrosinase-type molecule.

Effects of recombinant human interleukin-6 alone and in combination with recombinant interleukin-1 alpha and tumor necrosis factor alpha on antibacterial resistance in mice.

In this study, recombinant human interleukin-6 (rIL-6) was tested for its ability to alter the resistance of mice to experimental Listeria monocytogenes infection. Single bolus or repeated injections of rIL-6 by itself did not increase antilisteria resistance. When rIL-6 was injected in combination with suboptimal concentrations of rIL-1 alpha and tumor necrosis factor alpha (rTNF-alpha), it did not augment their abilities to mediate protection in the spleen and had a marginal effect on the level of protection in the liver. Injection of rIL-6 together with protective doses of rIL-1 alpha did not diminish the protection stimulated by the latter. Unlike rIL-1 alpha and recombinant tumor necrosis factor alpha, rIL-6 appears to have little ability to elevate antibacterial resistance.

Identification of regulatory Hck and PAI-2 proteins in the monocyte response to PEG-containing matrices

Mass spectrometry is a powerful proteomic tool enabling researchers to survey the global proteome of a cell. This technique has only recently been employed to investigate cell-material interactions. We had previously identified material scarcity and limited adherent cells as challenges facing mass spectrometric analysis of cell-material interactions. U937 adherent to tissue culture poly(styrene) was used as a model system for identifying proteins expressed by adherent monocytes and analyzed by HPLC coupled offline to MALDI-ToF/ToF (LC-MALDI). We identified 645 proteins from two cation fractions of crude U937 monocyte cell lysate. Forty three proteins of interest from the 645 were chosen based on literature searches for relevance to monocyte-material inflammation and wound healing. Proteins such as 40S ribosomal protein S19 and tyrosyl tRNA synthetase highlight the ability of LC-MALDI to identify proteins relevant to monocyte-material interactions that are currently unexplored. We used PEG-based semi-interpenetrating polymer networks and PEG-only hydrogels to investigate surface dependent effects on the Src family kinase Hck and plasminogen activator inhibitor-2 (PAI-2) using the pyrazolo pyrimidine small molecule inhibitor PP2 and exogenous urokinase plasminogen activator addition, respectively. Hck is well researched in cell adhesion while PAI-2 is virtually unknown in cell-material interactions. U937 on TCPS and PEG-only hydrogels secreted similar levels of inflammatory cytokines and gelatinase MMP-9. MCP-1 secretion from monocytes on PEG-only hydrogels was Hck independent in contrast to Hck-dependent MCP-1 secretion in U937 on TCPS. Overall, U937 adherent to sIPNs secrete low levels of soluble gelatinase MMP-9, IL-1beta, TNF-alpha, IL-6, and MCP-1 independent of Hck and PAI-2. This work demonstrates significant changes in surface dependent expression of proteins from monocytes adherent to PEG-based materials compared to TCPS.

2 In Vitro Analysis: 2.1 Isolation and Preparation of Lymphocytes from Infected Animals

Publisher Summary This chapter describes the isolation and preparation of lymphocytes from infected animals. Lymphocytes are the cells that provide specificity to host defense. Identifying the phenotype and antigen specificity of lymphocytes that have been isolated from animals infected with microbial agents is integral to understanding protective immunity. There are various methods of isolating, purifying, and characterizing lymphocytes from the tissues of infected animals. The unperturbed peritoneal cavity contains a mixed population of mononuclear cells. Neutrophils and eosinophils should be present in very low numbers. Mast cells are a minority population, but are conspicuous because of their large basophilic granules. Lymph node cell suspensions contain B and T lymphocytes, as well as macrophages, dendritic cells, and stromal cells. The numbers of lymphocytes recovered from lymph nodes depend on which nodes are collected and whether there has been recent antigen stimulation. Mononuclear phagocytes obtained from different tissue sites are all adherent, but can differ in how strongly they adhere. Cells activated at the sites of inflammation in vivo are more adherent than resident cells.

Isolation and preparation of lymphocytes from infected animals for in vitro analysis

Publisher Summary This chapter discusses the isolation and preparation of lymphocytes from infected animals for in vitro analysis. Lymphocytes are the cells that provide specificity to host defense. Identifying the phenotype and antigen specificity of lymphocytes that have been isolated from animals infected with microbial agents is integral to understand protective adaptive immunity. Lymphocytes can be obtained from a variety of the tissues of infected animals. The numbers of cells that can be recovered from each site vary depending on the type of tissue and the age and physiological status of animals. There are various methods to isolate, purify, and characterize lymphocytes from the tissues of infected animals. Some of these are elegant and sophisticated procedures that rely on expensive instrumentation to yield highly purified and well-characterized cell populations. The chapter focuses largely on simple preparative techniques that can be used by nearly any microbiology laboratory. These techniques will yield populations of lymphocytes suitable for functional assessment in vitro or adoptive transfer to recipient animals in vivo. The chapter discusses the use of adherence to remove mononuclear phagocytes and the use of nylon wool to enrich for T lymphocytes.

Dissociation of macrophage cytolysis and ability to transfer anti-listeria resistance by Concanavalin A-stimulated spleen cells

In this study, we determined whether spleen cells from Listeria monocytogenes-immunized mice were cytolytic for Listeria-infected macrophages. Spleen cells freshly obtained from immunized donors were unable to lyse Listeria-infected macrophages unless they were first stimulated in vitro for 2-3 days with Concanavalin A (ConA) or L. monocytogenes. Spleen cells from non-immunized mice developed cytolytic activity after incubation with ConA, but not with L. monocytogenes. Cytolytic spleen cells demonstrated an equivalent ability to lyse uninfected and Listeria-infected thioglycollate elicited peritoneal macrophages. Maximal cytolysis required co-incubation of effector and target cells for 18-20 h. Spleen cell culture supernatants did not lyse macrophages, suggesting that cytolysis required direct contact. Preincubation of immune spleen cells with ConA decreased their ability to transfer anti-listeria resistance in the spleens, but not the livers of recipient mice. Depletion of CD4+ or CD8+ cells did not significantly reduce the ability of ConA-incubated Listeria-immune spleen cells to transfer resistance. Despite being cytolytic for Listeria-immune infected macrophages, ConA-stimulated non-immune spleen cells did not transfer anti-listeria resistance. These results indicate that cytolytic cells can be generated by short-term incubation of spleen cells with antigen or mitogen. The dissociation between in vitro cytolytic activity and ability to transfer protection, however, suggests that the two biological activities are not inextricably linked.