R T Gazzinelli

Identification of Toxoplasma gondii in Paraffin-Embedded Sections by the Polymerase Chain Reaction

We used the polymerase chain reaction to amplify DNA fragments specific to Toxoplasma gondii. The sensitivity of the technique allowed for the detection of as few as ten cultured T. gondii tachyzoites. We applied the same amplification technique to deparaffinized ocular sections from two cases of ocular toxoplasmosis. Although toxoplasmic cysts could only be seen in one eye by optical microscopy, polymerase chain reaction allowed the identification of the parasite in both cases. Our study indicates the feasibility of a sensitive DNA-based assay to complement pathologic studies of an ocular parasitic disease.

CD8+ T-cell interactions with Toxoplasma gondii: implications for processing of antigen for class-I-restricted recognition

CD8+ T lymphocytes contribute to the control of acute and chronic T. gondii infection, and the activity of these cells is closely related to IFN gamma production. However, it is not clear whether CD8+ lymphocytes mediate protection solely by production of IFN gamma, or whether the lymphokine synergizes with CD8+ CTL activity. Interestingly, sensitization of bone marrow macrophages for CTL lysis can occur either as a result of infection or incubation with soluble T. gondii antigen. Thus, this system offers a powerful approach for analysing antigen processing pathways employed by intracellular protozoa, as well as for identifying the peptide epitopes which serve as targets for CD8(+)-dependent immunity.

Retrovirus-elicited interleukin-12 and tumour necrosis factor-alpha as inducers of interferon-gamma-mediated pathology in mouse AIDS.

Spleen cells from mice resistant or sensitive to mouse acquired immune deficiency syndrome (MAIDS) were examined for cytokine mRNA. In MAIDS‐resistant BALB/c mice, cytokine transcripts peaked at 1 week after infection with Type 1 cytokines [interleukin‐2 (IL‐2), tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), IL‐12], dominating over Type 2 cytokines (IL‐4, IL‐10). Expression of cytokines other than IL‐2 later declined to levels seen in uninfected mice. In MAIDS‐sensitive B6 mice, transcripts for all cytokines were increased at 1 week and, except for IL‐2, increased progressively. Spontaneous production of IFN‐γ protein was associated with enhanced mRNA expression at 1 week after infection of either strain, but none was detectable in association with even higher levels of transcripts at later times after infection of B6 mice. Spleen cells from longer‐term‐infected B6 mice, however, produced substantial amounts of IFN‐γ following treatment with lipopolysaccharide (LPS) or IL‐12. Inclusion of anti‐IL‐12 or anti‐TNF‐α antibodies blocked induction of IFN‐γ by LPS. Induction of IFN‐γ by IL‐12 was potentiated by TNF‐α following stimulation of intact spleen cells and purified CD4+ or CD8+ T cells, as well as negatively selected CD4−8− cells from infected B6 mice. Further studies showed that IFN‐γ knockout mice on a B6 background developed MAIDS with a prolonged time–course, whereas BALB/c knockout mice were unchanged in their resistance to MAIDS. These studies suggest that continuing low‐level expression of IFN‐γ, stimulated by IL‐12 and TNF‐α, contributes to the susceptibility of B6 mice to MAIDS but is not required for the resistance of BALB/c mice to disease.

Cells and cytokines in the pathogenesis of MAIDS, a retrovirus-induced immunodeficiency syndrome of mice

ConclusionsMAIDS is a retrovirus-induced immunodeficiency syndrome of mice that develops as a consequence of multiple interactions between T and B cells. The disease develops in response to infection with a defective virus encoding a Pr60gag protein that is myristylated and binds to cell membranes. T cells, B cells, and macrophages are all targets for infection by the defective virus, and some direct effects of infection have been demonstrated for T cells in the absence of other cell types. It is not known if the defective virus has direct effects on B cells and macrophages. It is clear, however, that an unusually vigorous response of the immune system to the inciting virus elicits deregulated expression of numerous cytokines. This response is antigen driven, as it does not develop in mice lacking MHC class II molecules, but the nature of the antigenic molecule is not known. Other T cell-B cell interactions of major importance to induction of disease include those mediated by ligation of CD40/CD40L and CD11a/CD54. It would appear that a significant component of the abnormal response is due to stimulation of T cells through the TCR in the absence of efficient co-stimulation, resulting in generalized anergy. The pattern of cytokine expression in MAIDS-susceptible mice — high IL-4, IL-10, and IFN-γ and low IL2 — is consistent with this model. By further increasing IFN-γ expression through treatment of mice with IL-12, it is possible to inhibit induction of disease and to reverse many manifestation of established disease. Disease inhibition by IL-12 can be ascribed in part to effects in limiting expression of the etiological virus. IL-12 may prove to be an effective treatment for other retrovirus infections with similar immunopathogenesis.

Cytokines as determinants of disease and disease interactions

The immune response to pathogens results in both host resistance and immunopathology. Cytokines and in particular those lymphokines produced by Th1 and Th2 cells play a key role in determining the balance between these two immunologic outcomes. Recent data suggest that interleukin-10, a product of both Th2 cells and macrophages, protects the host against excessive immunopathology. The cytokine environment generated by different pathogens may also influence the course and outcome of infections with unrelated organisms. This relationship may be particularly important in the case of HIV-1 where prior Th1 or Th2 biases established by helminth or intracellular infections may influence either initial viral susceptibility or drive progression to AIDS through immune activation.

Role of IL12 in MAIDS

Cousens, LP, Orange, J.S. & Biron, CA. (1995), EndogeWysocka, M., Kubin, M., Vieria, L.Q., Ozmen, L., nous interleukin-2 (IL-2) contributes to T cell expanGarotta, G., Scott, P. & Trinchieri, G. (1995), Intersion and interferon y (IFN-y) production during leukin-12 is required for interferon-y production and lymphocytic choriomeningitis virus infection. J. lethality in lipopolysaccharide-induced shock in Immunol., 155, 5690-5699. mice. Eur. J. Immunol., 25, 672-676. Orange, J.S. & Biron, C.A. (1996). An absolute and restricted requirement for interleukin12 in natural killer (NK) cell interferon-y production and antiviral defense: studies of NK and T cell responses in contrasting viral infections, J. Zmmunol., 156 (in press). Orange, J.S., Salazar-Mather, T.P., Opal, S.M., Spencer, R.L., Miller, A.H., McEwen, B.S. & Biron, C.A. (1995), Mechanism of interleukin 1Zmediated toxicities during experimental viral infections: role of tumor necrosis factor and glucocorticoids. J. Exp. Med., 181, 901-914.

Protocol for Assaying CTL Activity Against Toxoplasma gondii

Tachyzoites of the ts-4 strain of T. gondii are maintained by in vitro passage in human foreskin fibroblasts at 33°C. T. gondii soluble antigens (STAg) are prepared from parasites obtained by peritoneal lavage, 3 days after infection of Swiss Webster mice with 5–10 × 106 RH strain tachyzoites. Tachyzoites are concentrated by centrifugation (1500 × g, 10 min) and repeatedly sonicated until no intact parasites remain. After centrifugation at 10,000 × g for 30 min, the supernatant is collected, dialyzed against PBS, and stored at −80°C.

Immunologic Control of Toxoplasma gondii Infection by CD8+ Lymphocytes: A Model for Class I MHC-Restricted Recognition of Intracellular Parasites

Immunity against parasites (protozoa and helminths) has traditionally been thought to depend exclusively on CD4+ helper functions controlling B-cell (i.e., antibody) and lymphokine-mediated effector mechanisms. It is only within the last decade that CD8+ lymphocytes have been demonstrated to display protective activity against parasitic infections. As would be predicted, most CD8+-dependent effector mechanisms are directed against intracellular parasites infecting host cells expressing class I major histocompatibility complex (MHC) products. Nevertheless, there are several examples of CD8+ cells interacting with extra-cellular parasites such as tachyzoites of Toxoplasma gondii (Khan et al., 1988) or schistosomula of Schistosoma mansoni (Butterworth et al., 1979). In the latter system, recognition is dependent on the presence of adsorbed MHC molecules on the parasite surface (Sher et al., 1978). While most examples of CD8+-dependent killing are directed against protozoa parasitizing host cells, the intracellular environments of the affected organisms differ considerably. Because of this, the study of parasite-CD8+ interactions provides some fascinating as well as highly relevant models for studying processing and presentation of foreign antigens by class I molecules.

Synergistic and Compensatory Roles of CD4+, CD8+ T-Lymphocytes and Natural Killer Cells in Resistance to Toxoplasma Gondii

Cell-mediated immunity has long been recognized to play a crucial role in resistance to Toxoplasma gondii. Nevertheless, it is only within recent years that the events involved in cellular control of acute and chronic infection have begun to be defined. An important breakthrough occurred when Suzuki and Remington [Suzuki et al 1988] discovered the central role played by IFN- γ in resistance to new infection as well as in the control of re-activation. While the key effector function of this cytokine has now been confirmed, its mechanism of action remains poorly understood.