The potential of the dense granule antigens GRA1 and GRA6 of Toxoplasma gondii to be used as diagnosis reagents in a recombinant form was evaluated. Both proteins were expressed in Escherichia coli as glutathione-S-transferase (GST) fusions. The GST-GRA1 fusion comprises the entire GRA1 sequence devoid of its N-terminal signal peptide. Separate expression of the two N-and C-terminal hydrophilic regions of GRA6 showed that only the N-terminal hydrophilic part of the protein was recognized by a pool of positive human sera in an immunoblot. One hundred T. gondii-positive and 98 negative human sera were tested in two separate immunoglobulin G (IgG)-direct enzyme-linked immunosorbent assays (ELISAs) using either GST-GRA1 or GST-GRA6-Nt recombinant protein. Whereas the sensitivity of the GST-GRA1 IgG ELISA was low (68%), the GST-GRA6-Nt IgG ELISA reached a sensitivity of 96%. The reactivity to GRA6-Nt was shown to be high even with human sera of low IgG titers. In addition, comparison of the optical density values for each serum revealed that GRA1 may complement GRA6-Nt to reach an overall sensitivity of 98%. Therefore, the GST-GRA6-Nt ELISA could be used together with another antigen like GRA1 for the development of a recombinant antigenbased test for serodiagnosis of toxoplasmosis.Diagnosis of Toxoplasma gondii infections is of great medical importance for humans, especially pregnant women and immunosuppressed patients. During pregnancy, primary infection of women is often associated with fetal infection, which can lead to abortion or severe neonatal malformations. In immunocompromised adults (e.g., AIDS patients), toxoplasmosis (acute or, most often, reactivation of chronic infection) frequently causes a life-threatening encephalitis (for a review, see reference 12).Among the available diagnostic tests, serology is commonly used. Despite the fact that serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. At present, the detection of specific antibodies based on the recognition of crude Toxoplasma antigens requires mass production of the parasite either from peritoneal fluids of infected mice or from tissue cultures. The use of an Escherichia coli recombinant antigen(s) would be greatly beneficial in improving standardization of the tests and reducing their production cost.Enzyme-linked immunosorbent assay (ELISA) tests using Toxoplasma recombinant antigens have already been reported (4,9,10,11,14,17), but compared to the current serological tests, none of these recombinant antigens has allowed detection of all serologically positive individuals. It has emerged from these studies that the use of two or several recombinant antigens could be necessary to improve the sensitivity of these ELISA tests. Our previous studies on the secreted dense granule (GRA) antigens have shown that the recombinant forms of GRA1 (1), GRA2 (8), and GRA6 (this paper) are recognized by immune human sera. Here we report expression of both the GRA1 and GRA6 proteins in fusion with gl...

Emergency monocytopoiesis is an inflammation-driven hematological process that supplies the periphery with monocytes and subsequently with macrophages and monocyte-derived dendritic cells. Yet, the regulatory mechanisms by which early bone marrow myeloid progenitors commit to monocyte-derived phagocytes during endotoxemia remains elusive. Herein, we show that type I interferons signaling promotes the differentiation of monocyte-derived phagocytes at the level of their progenitors during a mouse model of endotoxemia. In this model, we characterized early changes in the numbers of conventional dendritic cells, monocyte-derived antigen-presenting cells and their respective precursors. While loss of caspase-1/11 failed to impair a shift toward monocytopoiesis, we observed sustained type-I-IFN-dependent monocyte progenitors differentiation in the bone marrow correlated to an accumulation of Mo-APCs in the spleen. Importantly, IFN-alpha and -beta were found to efficiently generate the development of monocyte-derived antigen-presenting cells while having no impact on the precursor activity of conventional dendritic cells. Consistently, the LPS-driven decrease of conventional dendritic cells and their direct precursor occurred independently of type-I-IFN signaling in vivo. Our characterization of early changes in mononuclear phagocytes and their dependency on type I IFN signaling during sepsis opens the way to the development of treatments for limiting the immunosuppressive state associated with sepsis.

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