An in vitro Absorption Model System Based on Cell Monolayers (Intestine 407, MDBK)

ABSTRACT Most Bacillus cereus toxin production is controlled by the quorum-sensing-dependent, pleiotropic global regulator plcR, which contributes to the organism's virulence in the eye. The purpose of this study was to analyze the effects of B. cereus infection and plcR-regulated toxins on the barrier function of retinal pigment epithelium (RPE) cells, the primary cells of the blood-retina barrier. Human ARPE-19 cells were apically inoculated with wild-type or quorum-sensing-deficient B. cereus, and cytotoxicity was analyzed. plcR-regulated toxins were not required for B. cereus-induced RPE cytotoxicity, but these toxins did increase the rate of cell death, primarily by necrosis. B. cereus infection of polarized RPE cell monolayers resulted in increased barrier permeability, independent of plcR-regulated toxins. Loss of both occludin and ZO-1 expression occurred by 8 h postinfection, but alterations in tight junctions appeared to precede cytotoxicity. Of the several proinflammatory cytokines analyzed, only interleukin-6 was produced in response to B. cereus infection. These results demonstrate the deleterious effects of B. cereus infection on RPE barrier function and suggest that plcR-regulated toxins may not contribute significantly to RPE barrier permeability during infection.

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Specific elimination of cytotoxic effector cells. I. Adsorptive behavior of effectors and their precursors and spleen cell monolayers.

Journal of Experimental Medicine ◽

10.1084/jem.144.4.996 ◽

1976 ◽

Vol 144 (4) ◽

pp. 996-1008 ◽

Cited By ~ 16

Author(s):

J R Neefe ◽

D H Sachs

Keyword(s):

Mouse Spleen ◽

Spleen Cells ◽

Effector Cells ◽

Cell Monolayers ◽

Specific Suppression ◽

Normal Spleen ◽

Adsorptive Behavior ◽

Cytotoxic Effector ◽

Primed Mouse

Monolayers formed of normal mouse spleen cells attached to polystyrene coated with poly-L-lysine were tested for their ability to bind specifically antigen-reactive cells in normal or primed mouse spleen. 88 to greater than 98% of the activity of cytotoxic populations was removed by a single adsorption. However, normal spleen cells or spleen cells previously primed in vitro could not be depleted of their capacity to be sensitized, even when adsorption effectively removed all residual cytotoxic activity from the same previously primed population. In fact, exposure to an immunoadsorbent augmented the ultimate cytotoxicity generated in a nonspecific fashion. This augmentation was especially dramatic in the case of a previously primed population and may have reflected the removal of a nonspecific suppressor. If antigen-reactive precursors cannot be removed efficiently by adsorption, other approaches to the generation of tolerant lymphoid populations, such as specific suppression of precursor differentiation must be sought.

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Exposure of ichnovirus particles to digitonin leads to enhanced infectivity and induces fusion from without in an in vitro model system

Journal of General Virology ◽

10.1099/vir.0.83118-0 ◽

2007 ◽

Vol 88 (11) ◽

pp. 2977-2984 ◽

Cited By ~ 4

Author(s):

Don Stoltz ◽

Renée Lapointe ◽

Andrea Makkay ◽

Michel Cusson

Keyword(s):

Outer Membrane ◽

In Vitro Model ◽

Model System ◽

Host Cells ◽

Fusion Event ◽

Susceptible Host ◽

In Vitro Model System ◽

Vitro Model

Unlike most viruses, the mature ichnovirus particle possesses two unit membrane envelopes. Following loss of the outer membrane in vivo, nucleocapsids are believed to gain entry into the cytosol via a membrane fusion event involving the inner membrane and the plasma membrane of susceptible host cells; accordingly, experimentally induced damage to the outer membrane might be expected to increase infectivity. Here, in an attempt to develop an in vitro model system for studying ichnovirus infection, we show that digitonin-induced disruption of the virion outer membrane not only increases infectivity, but also uncovers an activity not previously associated with any polydnavirus: fusion from without.

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CP30, a Cysteine Proteinase Involved inTrichomonas vaginalis Cytoadherence

Infection and Immunity ◽

10.1128/iai.68.9.4907-4912.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 4907-4912 ◽

Cited By ~ 82

Author(s):

M. Remedios Mendoza-López ◽

Cecilia Becerril-Garcia ◽

Loriz V. Fattel-Facenda ◽

Leticia Avila-Gonzalez ◽

Martha E. Ruíz-Tachiquín ◽

...

Keyword(s):

Epithelial Cells ◽

Hela Cell ◽

Trichomonas Vaginalis ◽

Cysteine Proteinase ◽

Collagen Iv ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cell Monolayers ◽

Thiol Proteinase

ABSTRACT We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.

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