Annie Bonhomme

Value of specific immunoglobulin A detection by two immunocapture assays in the diagnosis of toxoplasmosis.

The diagnosis ofToxoplasma gondii infection is currently based on immunological tests, but tests for IgG and IgM antibodies alone are often insufficient to assess the risk of active disease, especially during pregnancy and in immunodeficient subjects. The supplementary diagnostic value of testing for antitoxoplasmic IgA in cases of acute, chronic, congenital and reactivated toxoplasmosis, relative to classical immunological tests, was evaluated using two immunocapture tests, one based on tachyzoite agglutination and the other on an immunoenzymatic complex recognizing the membrane protein P30 ofToxoplasma gondii. A total of 4,541 sera from 395 uninfected subjects, 468 immunized subjects with chronic infection, 117 subjects with acute infection and 403 children, 103 of whom had congenital toxoplasmosis, was tested. Specific IgA tests were negative in the nonimmune population, but tests for this immunoglobulin subtype became positive very rapidly during primary infection, and IgA disappeared more rapidly than IgM. In the children infected in utero, specific IgA was detected more frequently than IgM. In contrast, in a population of HIV-seropositive subjects with clinical toxoplasmosis, tests for IgA were poorly sensitive. The two tests for specific IgA produced similar results, except in the early stages of primary infection, in which immunoenzymatic testing for anti-P30 IgA was less sensitive than the agglutination method.

Tachyzoite calcium changes during cell invasion by Toxoplasma gondii

Abstract The invasion of host cells by the obligate intracellular protozoan parasite Toxoplasma gondii is calcium dependent. We have identified two calcium storage areas in tachyzoites, the endoplasmic reticulum and vesicles that contain high concentrations of calcium as amorphous calcium phosphate precipitates. Our data indicate that these vesicles slowly lose their calcium during the intracellular development of the tachyzoite as their nucleus phosphorus content increases. We found fluctuations in the sulfur content of the tachyzoite during invasion following the exocytosis of protein from the secretory organelles, with a loss of sodium and chlorine, and the uptake of potassium from the host cell cytoplasm. We demonstrated that penetration of the tachyzoite into the host cell was accompanied by increases in the concentrations of phosphorus and sulfur in the host cell nucleus, probably due to increased transcription. The cytosol sodium concentrations decreased, while the potassium content increased. Thus, the subcellular element distribution of tachyzoites and host cells changes during invasion and intracellular growth of the parasites. In addition, our results indicate that tachyzoite calcium might be involved in the egress of the parasite from the host cell.

Involvement of tumor necrosis factor-α during infection of human monocytic cells by Toxoplasma gondii

Abstract Although the involvement of tumor necrosis factor-α (TNF-α) during Toxoplasma gondii infection has largely been described in mouse models, only a few studies in human models have been reported. We demonstrated the role of TNF-α during the process of invasion by T. gondii in human monocytic cells. Cotreatment of cells with interferon-γ (IFN-γ) and lipolysaccharide (LPS) during T. gondii infection induced TNF-α production and decreased the number of parasitized cells (invasion index). Neutralization of the production of TNF-α using a specific monoclonal antibody or pentoxifylline enhanced the invasion index. The relationship between TNF-α production and protection of monocytic cells against T. gondii invasion was confirmed by treatment of infected cultures with exogenous TNF-α. Thus, in contrast to the results obtained in murine models but in accordance with those observed in other human models, the present study shows for the first time in human monocytic cells that T. gondii does not induce any TNF-α secretion and inhibits TNF-α production induced by IFN-γ/LPS.

Does human toxoplasmosis involve an imbalance in T1/T2 cytokines?

The T1 (interferon-gamma, interleukin-12, interleukin-2) and T2 (interleukin-4, interleukin-10, interleukin-6) cytokine groups constitute two polar responses of the immune system. The T1 group is a predominantly cellular response, while the T2 group response is mainly humoral. The hypothesis forwarded here links these subgroups of induced cytokines to the various clinical forms of human toxoplasmosis. Ocular toxoplasmosis in immunocompetent patients could be attributed to a T1 hyper-response, whereas congenital toxoplasmosis, toxoplasmic encephalitis (in immunodeficient patients) and active chronic toxoplasmosis (with persistent lymphadenophathy) would be characterized by a predominantly T2 response. Confirmation that this kind of immunological imbalance effectively underlies the various clinical forms of toxoplasmosis would open the way for a new range of treatments based on immunomodulation.

Interferon-gamma signal transduction during parasite infection: modulation of MAP kinases in the infection of human monocyte cells (THP1) by Toxoplasma gondii

We assayed mitogen‐activated protein (MAP) kinase phosphorylation in a human monocyte cell line (THP1) during their infection by Toxoplasma gondii. In addition, we tested the effect of specific MAP kinase inhibitors (PD098059 and SB203580) on parasite invasion. MAP kinase phosphorylation was increased in the cytosol and membrane fractions of THP1 infected with T. gondii. The MAP kinase phosphorylation of uninfected THP1 cells was not significantly modified by incubation for 20 h with 1000 U/ml of IFN‐γ. However, IFN‐γ treatment of infected cells significantly reduces the increase in phosphorylation caused by parasite infection. There was also MAP kinase activity in the cytosol and membrane fractions of extracellular T. gondii tachyzoites. IFN‐γ altered the distribution of activity in subcellular fractions of extracellular T. gondii tachyzoites. This indicates that IFN‐γ directly affects parasite MAP kinase activity. The results provide evidence that MAP kinase pathways participate in the infection by T. gondii and that the decrease in MAP kinase activity in infected cells caused by IFN‐γ may be involved in mediating their protective signals.

Regulation of tumor necrosis factor alpha and its specific receptors during Toxoplasma gondii infection in human monocytic cells

Abstract. The coccidian Toxoplasma gondii is an obligate intracellular parasite which can infect all cell types. Among the cytokines elicited by the immune response to Toxoplasma, tumor necrosis factor alpha (TNF-α) acts synergistically with gamma interferon (IFN-γ) and plays a major role in host cell resistance. We have previously reported that TNF-α production induced by IFN-γ/LPS decreases after T. gondii infection of human myelomonocytic THP-1 cells. Here, we investigated the regulation of TNF-α and its specific receptors during T. gondii infection of THP-1 cells. We found that TNF-α production was regulated at a post-transcriptional level and that TNF receptor expression was regulated at a pretranscriptional level. The TNF receptor I shedding and the fall in TNF-α levels observed after T. gondii infection would thus be induced by a parasite component with serine protease activity. These findings indicate that T. gondii participates not only in controlling the cytotoxic effects of TNF-α during the infection process, but also in signal transduction mediated mainly by TNF receptor I.

Toxoplasma gondii: Immunocytochemistry of four immunodominant antigens with monoclonal antibodies

Four monoclonal antibodies in which diagnostic usefulness has been observed, concerning congenital, acquired, and reactivated toxoplasmosis, were raised against Toxoplasma gondii tachyzoïtes in order to localize immunodominant antigens. On immunoblots, it appears that McAb IV47, McAB GII9, McAb II38, and McAb IE10 identify families of proteins with estimated molecular weights of 28-30 kDa, 30 kDa, 45-50 kDa, and 66-70 kDa, respectively. By immunogold preembedding techniques one can observe an homogeneous labeling of the outer pellicle of the tachyzoïtes with the McAb GII9 and IV47 and a light labeling with the McAb II38 and IE10. The three-dimensional observation of cell surface antigens is performed by applying a modified metal extraction replica method, i.e., A plasma polymerization method of glow discharge by Tanaka (1979). By immunogold preembedding techniques [with saponin permeabilization (0.1%)], and by immunogold postembedding techniques, a labeling of the rhoptries is observed with McAb GII9 and McAb IV47 but essentially all label is found with McAb II38 and IE10. With McAb GII9 a uniform labeling is observed on the cell surface. By immunoenzymatic techniques (peroxidase) a cell surface labeling is observed with the four McAb. Intracellular Toxoplasma, the outer pellicle, and the vesicles of the network (elaborated by Toxoplasma in parasitophorous vacuole) are also labeled with McAb IE10. These results indicate that McAb GII9 recognizes antigens of the antigen family (P 30) located on the cell surface and in the rhoptries. The antigen recognized by McAb IV47 is essentially located on and beneath the Toxoplasma cell surface membrane, and McAb II38 and IE10 identify preferentially rhoptry proteins.

Quantitative immunolocalization of four immunodominant antigens of Toxoplasma gondii

Summary— Ultrastructural localization of four immunodominant antigens of Toxoplasma gondii was investigated quantitatively on thin sections and replicas by an immunogold technique using four monoclonal antibodies (Mab). On immunoblot Mab IV47, GII9, II38 and IE10 identified proteins of 28, 30, 45 and 66–70 kDa, respectively. Use of digital image analyzer and a semi‐automatic procedure developed by us, the patterns of label distribution were compared in three cell structures: cell surface, submembrane area and rhoptries. On the whole cell surface, protein P28 and P30 were 2.5 and 4 times more abundant than P66–70 respectively. The protein P28 was essentially concentrated in the submembrane area with a labeling of 195.4 ± 46.7 gold particles/μm2 that follows a decreasing gradient from this area to the cell centre. In the rhoptries, all four antigens were detected, P45 and P66–70 being major with a labeling of 97.1 ± 31.1 gold particles/μm2 and 155.1 ± 39.3 gold particles/μm2 respectively. The results support the hypothesis that rhoptries are the essential site for antigen storage.

Kinetics study of the localization and quantitation of target antigens of immunoglobulin a antibodies in acquired and congenital toxoplasmosis

The cellular distribution (localization and quantitation) of the target parasite’s antigens in the tachyzoite along the IgA kinetics was determined in the course of acquired toxoplasmosis and congenital toxoplasmosis. In the case of acquired toxoplasmosis, throughout the IgA kinetics a correlation was noted between the membrane and submembrane immunolabeling and the results of the immunocapture and enzyme-linked immunosorbent assay IgA (ELISA-A) tests. The rhop-tries’ immunolabeling remained higher. The immunolabeling evolution and the results of the immunology tests were not closely related to the treatment (Rovamycin). From the congenital toxoplasmosis cases it was observed that membrane immunolabeling correlated with the results of the serology tests and with the treatment (Fansi-dar). The rhoptry antigens were recognized throughout the IgA kinetics; even when the serology tests became negative, immunolabeling persisted. Rhoptries appeared as secretory organelles of antigens recognized during acute, chronic, and congenital stages of Toxoplasma infection.

Une famille d'enzymes présentes des protozoaires aux mammifères: comment les phospholipases A2 participent au processus d'invasion de Toxoplasma gondii

Abstract Toxoplasma gondii is an intracellular obligate protozoan parasite. Human infection is generally subclinical but hosts with defective cellular immunity are at risk of severe disease. In many countries, congenital toxoplasmosis and toxoplasmic encephalitis in HIV-infected individuals are significant causes of morbidity and mortality. We review here the role of the members of phospholipases A2 (PLA2) family and how they participate in the invasion process of T. gondii. PLA2 have been described in mammals cells as a family composed of nine groups of enzymes that specifically hydrolyse sn-2 bonds of phospholipids. Each PLA2 group have a distinctive substrate preference, localization and way of activation indicating different physiological roles. We describe the existence of three PLA2 isoforms in T. gondii. Inhibitors of secretory PLA2 isoforms (sPLA2) and cytosolic PLA2 (cPLA2), showed that cell and parasite sPLA2 and parasite cPLA2, but not cell cPLA2, favours T. gondii invasion. The addition of IFNγ to cultured infected THP1 cells protected against T. gondii infection by an early mechanism involving a reduction in the number of parasitized cells. The reduction in the percentage of parasitized cells obtained by treatment with IFN γ is linked with a decrease in parasite and cellular PLA2 activity. This is a new effector mechanism of IFN γ against T. gondii infection. The inhibitors of sPLA2 type II have a pharmacological potential against T. gondii infection that remain to be tested in vivo.