Alkaline hydrolysis of mouse-adapted scrapie for inactivation and disposal of prion-positive material.

Prion diseases such as bovine spongiform encephalopathy, chronic wasting disease, and scrapie pose serious risks to human and animal health due to a host of disease-specific factors, including the resistance of infectious prions (PrP(Sc)) to natural degradation and to most commercial inactivation procedures. In an attempt to address this concern, a mouse model was used to compare the efficacy of an alkaline hydrolysis process with a simulated continuous-flow rendering treatment for disposal of PrP(Sc)-infected biological material. Female C57/BL6 mice (N = 120) were randomly divided into 4 treatment groups (n = 30), and each mouse was injected intraperitoneally with their designated treatment inoculum. Treatment groups 1 and 2 served as the positive and negative controls, respectively. Group 3 was inoculated with rendered scrapie-positive mouse brain material to investigate the effectiveness of simulated continuous-flow rendering practices to reduce or eliminate PrP(Sc). Group 4 was inoculated with hydrolyzed scrapie-positive mouse brain material to determine the sterilizing effect of alkaline hydrolysis on PrP(Sc). Mice were monitored for overt signs of disease, and those showing clinical signs were killed to prevent undue suffering. Brains were obtained from all mice that died (or were killed) and analyzed with an ELISA for the presence of PrP(Sc). Results indicated that the simulated continuous-flow rendering treatment used for preparing the rendering treatment group inoculum failed to completely eliminate PrP(Sc). Rendering delayed, but did not stop, clinical mouse-adapted scrapie transmission. Compared with positive controls, the rendering treatment group experienced an approximate 45-d average delay in days to death (250 vs. 205 d for positive controls; P < 0.0001) and a death loss of 73.9% (P = 0.0094). Positive controls suffered 100% death loss. The results validated the efficacy of the alkaline hydrolysis treatment to inactivate all PrP(Sc) because no alkaline hydrolysis treatment group mice succumbed to the disease (P < 0.0001). Based on our results, alkaline hydrolysis should be considered by the animal rendering and beef packing industries as an alternative to incineration, landfill burial, and rendering for disposing of biological material potentially infected or contaminated with prion disease.

Selection and characterization of Toxoplasma gondii mutants resistant to artemisinin.

Toxoplasma gondii infection, like malaria, is sensitive to inhibition by artemisinin (ART). Mechanisms of action for ART in malaria treatment have been proposed, but little is known about its effects in T. gondii infection. To better understand its inhibitory effects on T. gondii, mutants resistant to ART were selected by progressive culture in permissive levels of the drug. Five clonal isolates were established and characterized. The isolates were approximately 65-fold less sensitive to ART than is the parental RH and showed cross-resistance to the ART derivatives dihydroartemisinin and artemether. In addition to ART resistance, 1 clone (C9) formed morphologically unusual parasitophorous vacuoles and another (A2) was avirulent for mice and protected mice from challenge with the wild type. These clonal T. gondii mutant isolates will be useful for the study of not only the mechanism of action of ART but also parasite vacuole biology and virulence factors.

Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis.

Infection with certain intracellular pathogens, including viruses and bacteria, may induce host cell apoptosis. On the other hand, infection with some viruses inhibits apoptosis. Complex protozoan parasites, including Toxoplasma gondii and members of Plasmodium, Leishmania, and Microsporidia, are also obligate intracellular pathogens, yet relatively little is known regarding their subversion of host cell functions. We now report that cells infected with T. gondii are resistant to multiple inducers of apoptosis, including Fas-dependent and Fas-independent CTL-mediated cytotoxicity, IL-2 deprivation, gamma irradiation, UV irradiation, and the calcium ionophore beauvericin. Inhibition of such a broad array of apoptosis inducers suggests that a mechanism common to many, or perhaps all, apoptotic pathways is involved. The inhibitory activity requires live intracellular parasite and ongoing protein synthesis. Despite T. gondii-mediated inhibition of DNA fragmentation, infected cells can still be lysed by CTL.

CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans.

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.

Cytolysis mediated by ionophores and pore-forming agents: role of intracellular calcium in apoptosis.

Apoptosis is a term used to describe certain forms of physiological cell death that occur during embryogenesis, differentiation, and normal cell turnover. Previous reports concerning the effects of calcium ionophores on rodent thymocytes and the pore-forming proteins perforin and staphylococcal alpha-toxin on murine tumor cells led to the suggestion that simply raising intracellular calcium causes apoptotic cell death. This hypothesis was tested using two ionophores, A23187 and valinomycin, and two pore-forming agents, melittin and staphylococcal alpha-toxin, on four murine tumor cell lines. Although treatment with these agents could raise intracellular calcium, and in some instances cause DNA fragmentation, only valinomycin caused apoptosis. In contrast to previous reports, our results suggest that raising intracellular calcium and inducing internucleosomal DNA fragmentation is not sufficient to elicit apoptotic cell death in all cell types.