Identification of novel antigens for serum IgG diagnosis of human toxoplasmosis.

In the present study, we attempted to identify antigens with high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. We investigated soluble proteins from the tachyzoites of the RH strain of Toxoplasma gondii (T. gondii) and excreted/secreted antigens (ESAs) from the peritoneal protein of T. gondii-infected mice. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis revealed that in both soluble tachyzoite antigens and ESAs, the antigens located between 25 and 35 kDa had high diagnostic sensitivity. Further analysis of antigenic specificity revealed that the antigens located between 25 and 35 kDa were specifically recognized by the sera of toxoplasmosis patients, but other parasitic diseases were not. The protein spots between 25 and 35 kDa were selected after two-dimensional electrophoresis of both soluble tachyzoite antigens and ESAs. GRA2, GRA7, and triosephosphate isomerase (TPI) were successfully characterized from the protein spots using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis. We expressed, purified, and evaluated proteins GRA2, GRA7, and TPI. TPI is a novel antigen with potential for the serological diagnosis of toxoplasmosis, and composite recombinant proteins (TPI, GRA2, and GRA7) have great sera diagnostic value for the detection of the disorder.

In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

Triosephosphate isomerase of Taenia solium (TTPI): phage display and antibodies as tools for finding target regions to inhibit catalytic activity.

Previous studies demonstrated that antibodies against triosephosphate isomerase of Taenia solium (TTPI) can alter its enzymatic catalysis. In the present study, we used antibodies produced against the NH2-terminal region of TTPI (1/3NH2TTPI) and the phage display technology to find target regions to inhibit TTPI activity. As a first step, we obtained polyclonal antibodies against non-conserved regions from the 1/3NH2TTPI, which had an inhibitory effect of about 74 % on catalytic activity. Afterward, they were used to screen a library of phage-displayed dodecapeptides; as a result, 41 phage mimotope clones were isolated and grouped according to their amino acid sequence, finding the consensus A1 (VPTXPI), A2 (VPTXXI), B (LTPGQ), and D (DPLPR). Antibodies against selected phage mimotope clones were obtained by rabbit's immunization; these ones clearly recognized TTPI by both Western blot and ELISA. However, only the mimotope PDTS16 (DSVTPTSVMAVA) clone, which belongs to the VPTXXI consensus, raised antibodies capable of inhibiting the TTPI catalytic activity in 45 %. Anti-PDTS16 antibodies were confronted to several synthetic peptides that encompass the 1/3NH2TTPI, and they only recognized three, which share the motif FDTLQK belonging to the helix-α1 in TTPI. This suggests that this motif is the main part of the epitope recognized by anti-PDTS16 antibodies and revealed its importance for TTPI catalysis.

Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.

Inhibition of glyceraldehyde-3-phosphate dehydrogenase activity by antibodies present in the cerebrospinal fluid of patients with multiple sclerosis.

We have previously shown that B cells and Abs reactive with GAPDH and antitriosephosphate isomerase (TPI) are present in lesions and cerebrospinal fluid (CSF) in multiple sclerosis (MS). In the current study, we studied the effect of anti-GAPDH and anti-TPI CSF IgG on the glycolytic enzyme activity of GAPDH and TPI after exposure to intrathecal IgG from 10 patients with MS and 34 patients with other neurologic diseases. The degree of inhibition of GAPDH activity by CSF anti-GAPDH IgG in the seven MS samples tested varied from 13 to 98%, which seemed to correlate with the percentage of anti-GAPDH IgG in the CSF IgG (1-45%). Inhibition of GAPDH activity (18 and 23%) by CSF IgG was seen in two of the 34 patients with other neurologic diseases, corresponding to the low percentage of CSF anti-GAPDH IgG (1 and 8%). In addition, depletion of anti-GAPDH IgG from CSF IgG, using immobilized GAPDH, removed the inhibitory effect of the IgG on GAPDH. No inhibition of GAPDH activity was seen with CSF samples not containing anti-GAPDH IgG. No inhibition of TPI activity was seen with any purified CSF IgG sample. These findings demonstrate an increased percentage of anti-GAPDH Abs in the CSF of patients with MS that can inhibit GAPDH glycolytic enzyme activity and may contribute to neuroaxonal degeneration.

Inhibition of enzyme activity of Rhipicephalus (Boophilus) microplus triosephosphate isomerase and BME26 cell growth by monoclonal antibodies.

In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition  by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.

Glycolysis- and immune-related novel prognostic biomarkers of Ewing's sarcoma: glucuronic acid epimerase and triosephosphate isomerase 1.

INTRODUCTION: Owing to the poor prognosis of Ewing's sarcoma, reliable prognostic biomarkers are highly warranted for clinical diagnosis of the disease. MATERIALS AND METHODS: A combination of the weighted correlation network analysis and differentially expression analysis was used for initial screening; glycolysis-related genes were extracted and subjected to univariate Cox, LASSO regression, and multivariate Cox analyses to construct prognostic models. The immune cell composition of each sample was analysed using CIBERSORT software. Immunohistochemical analysis was performed for assessing the differential expression of modelled genes in Ewing's sarcoma and paraneoplastic tissues. RESULTS: A logistic regression model constructed for the prognosis of Ewing's sarcoma exhibited that the patient survival rate in the high-risk group is much lower than in the low-risk group. CIBERSORT analysis exhibited a strong correlation of Ewing's sarcoma with naïve B cells, CD8+ T cells, activated NK cells, and M0 macrophages (P < 0.05). Immunohistochemical analysis confirmed the study findings. CONCLUSIONS: GLCE and TPI1 can be used as prognostic biomarkers to predict the prognosis of Ewing's sarcoma, and a close association of Ewing's sarcoma with naïve B cells, CD8+ T cells, activated NK cells, and M0 macrophages provides a novel approach to the disease immunotherapy.

A Chimera of Th1 Stimulatory Proteins of Leishmania donovani Offers Moderate Immunotherapeutic Efficacy with a Th1-Inclined Immune Response against Visceral Leishmaniasis.

Immunotherapy, a treatment based on host immune system activation, has been shown to provide a substitute for marginally effective conventional chemotherapy in controlling visceral leishmaniasis (VL), the deadliest form of leishmaniasis. As the majority of endemic inhabitants exhibit either subclinical or asymptomatic infection which often develops into the active disease state, therapeutic intervention seems to be an important avenue for combating infections by stimulating the natural defense system of infected individuals. With this perspective, the present study focuses on two immunodominant Leishmania (L.) donovani antigens (triosephosphate isomerase and enolase) previously proved to be potent prophylactic VL vaccine candidates, for generating a recombinant chimeric antigen. This is based on the premise that in a heterogeneous population, a multivalent antigen vaccine would be required for an effective response against leishmaniasis (a complex parasitic disease). The resulting molecule rLdT-E chimeric protein was evaluated for its immunogenicity and immunotherapeutic efficacy. A Th1 stimulating adjuvant BCG was employed with the protein which showed a remarkable 70% inhibition of splenic parasitic multiplication positively correlated with boosted Th1 dominant immune response against lethal L. donovani challenge in hamsters as evidenced by high IFN-γ and TNF-α and low IL-10. In addition, immunological analysis of antibody subclass presented IgG2-based humoral response besides considerable delayed-type hypersensitivity and lymphocyte proliferative responses in rLdT-E/BCG-treated animals. Our observations indicate the potential of the chimera towards its candidature for an effective vaccine against Leishmania donovani infection.

Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.

Biochemical identification of a mutated human melanoma antigen recognized by CD4(+) T cells.

CD4(+) T cells play a critical role in generating and maintaining immune responses against pathogens and alloantigens, and evidence suggests an important role for them in antitumor immunity as well. Although major histocompatibility complex class II-restricted human CD4(+) T cells with specific antitumor reactivities have been described, no standard method exists for cloning the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spectrometry sequencing techniques and molecular cloning to isolate a unique melanoma Ag recognized by a CD4(+) tumor-infiltrating lymphocyte (TIL) line. The HLA-DRbeta1*0101-restricted Ag was determined to be a mutated glycolytic enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detected in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity was enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutated amino acid residue was a T cell receptor contact. Defining human tumor Ag recognized by T helper cells may provide important clues to designing more effective immunotherapies for cancer.

Triosephosphate isomerase, carbonic anhydrase, and creatinine kinase-brain isoform are possible antigen targets in patients with achalasia.

BACKGROUND: Idiopathic achalasia is an uncommon esophageal motor disorder. The disease involves interaction between inflammatory and autoimmune responses. However, the antigens related to the disease are still unknown. AIM: To identify the possible antigen targets in muscle biopsies from lower esophageal sphincter (LES) of achalasia patients. METHODS: Esophageal biopsies of patients with type I and type II achalasia and esophagogastric junction outflow obstruction (EGJOO) were analyzed. Lower esophageal sphincter muscle biopsy from a Healthy organ Donor (HD) was included as control for two-dimensional gel electrophoresis. Immunoblotting of muscle from LES lysate with sera of type I, type II achalasia, or type III achalasia, sera of EGJOO and sera of healthy subjects (HS) was performed. The target proteins of the serum were identified by mass spectrometry Matrix-assited laser desorption/ionization time-of-flight (MALDI-TOF). KEY RESULTS: The proteomic map of muscle from LES tissue lysates of type I, and type II achalasia, EGJOO, and HD were analyzed and divided into three important regions. We found a difference in the concentration of certain spots. Further, we observed the serum reactivity of type I achalasia and type II achalasia against 45 and 25 kDa bands of type I achalasia tissue. Serum of type III achalasia and EGJOO mainly recognized 25 kDa band. Bands correspond to triosephosphate isomerase (TPI) (25 kDa), carbonic anhydrase (CA) (25 kDa) and creatinine kinase-brain (CKB) isoform (45 kDa). CONCLUSIONS AND INFERENCES: We identify three antigen targets, TPI, CA, and CKB isoform, which are recognized by sera from patients with achalasia.

Autoantibodies associated with neuropsychiatric systemic lupus erythematosus: the quest for symptom-specific biomarkers.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs, including the central nervous system. Neuropsychiatric SLE (NPSLE) is a severe and potentially fatal condition. Several factors including autoantibodies have been implicated in the pathogenesis of NPSLE. However, definitive biomarkers of NPSLE are yet to be identified owing to the complexity of this disease. This is a major barrier to accurate and timely diagnosis of NPSLE. Studies have identified several autoantibodies associated with NPSLE;some of these autoantibodies are well investigated and regarded as symptom-specific. In this review, we discuss recent advances in our understanding of the manifestations and pathogenesis of NPSLE. In addition, we describe representative symptom-specific autoantibodies that are considered to be closely associated with the pathogenesis of NPSLE.

Detection of autoantibodies against cyclophilin A and triosephosphate isomerase in sera from breast cancer patients by proteomic analysis.

Much interest is presently being shown toward identifying markers for the detection of breast cancer. To detect autoantibodies that could represent diagnostic markers for breast cancer, we comprehensively analyzed serum autoantibodies showing immunoreactivity to proteins in tumor tissues of breast cancer. Tumor tissues were obtained from 40 patients with breast cancer, along with sera from 30 other patients with breast cancer and 22 healthy donors. Proteins from tumor tissues were separated by 2-DE. After blotting onto PVDF membranes, tissue proteins were immunoblotted with sera from patients or healthy donors. By comparing each immunoblot pattern, three immunoreactive spots displayed stronger staining intensity with patient sera than with sera from healthy donors. The matched protein spots on 2-DE gels were digested and used for LC-MS/MS analysis, and identified as cyclophilin A (peptidyl-prolyl cis-trans isomerase A), triosephosphate isomerase and ubiquitin-conjugating enzyme E2N. Immunoblot analysis was then performed using commercially available purified proteins, confirming the specificity of anti-cyclophilin A and anti-triosephosphate isomerase antibodies in sera from patients.

Identification of major allergens in watermelon.

BACKGROUND: Watermelon is a worldwide consumed Cucurbitaceae fruit that can elicit allergic reactions. However, the major allergens of watermelon are not known. The aim of this study is to identify and characterize major allergens in watermelon. METHODS: Twenty-three patients allergic to watermelon took part in the study. The diagnosis was based on a history of symptoms and positive skin prick-prick tests to watermelon, confirmed by positive open oral challenge testing to watermelon pulp. Allergenic components were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by N-terminal amino acid sequencing and mass spectrometry. Allergens were purified combining several chromatographic steps. RESULTS: Several IgE binding bands (8-120 kDa) were detected in watermelon extract. Three major allergens were identified as malate dehydrogenase (36 kDa), triose phosphate isomerase (28 kDa) and profilin (13 kDa). Purified allergens individually inhibited IgE binding to the whole watermelon extract. CONCLUSIONS: All in all these results indicate that malate dehydrogenase, triose phosphate isomerase and profilin are major allergens involved in watermelon allergy.

Cloning genes encoding MHC class II-restricted antigens: mutated CDC27 as a tumor antigen.

In an effort to identify tumor-specific antigens recognized by CD4(+) T cells, an approach was developed that allows the screening of an invariant chain-complementary DNA fusion library in a genetically engineered cell line expressing the essential components of the major histocompatibility complex (MHC) class II processing and presentation pathway. This led to the identification of a mutated form of human CDC27, which gave rise to an HLA-DR4-restricted melanoma antigen. A mutated form of triosephosphate isomerase, isolated by a biochemical method, was also identified as an HLA-DR1-restricted antigen. Thus, this approach may be generally applicable to the identification of antigens recognized by CD4(+) T cells, which could aid the development of strategies for the treatment of patients with cancer, autoimmune diseases, or infectious diseases.

[Enhancing protective immunity effects of TPI DNA vaccine against Schistosoma japonicum through codon optimization].

OBJECTIVE: To study the protective effect of codon optimized TPI DNA vaccine against Schistosoma japonicum infection. METHODS: Sixty female BALB/c mice were randomly divided into 5 groups. The mice were injected through musculus quadriceps femoris with 100 microg pcDNA 3.1 control (Group A), pcDNA3.1-TPI (Group B), pcDNA 3.1-TPI-mHSP70 (Group C), pcDNA3.1-TPI.opt (Group D), and pcDNA3.1-TPI.opt-mHSP70 (Group E) respectively. All mice were immunized for three times with an interval of two weeks. The mice were challenged with (40+/-1) cercariae of S. japonicum per mouse by abdominal skin penetration 4 weeks after the last immunization, and sacrificed at 42 days post-challenge, the number of worms or hepatic eggs was counted. Blood was taken for the detection of IgG, IgG1, and IgG2a 2 days before immunization and before challenge, respectively. Spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjCTPI peptide, and the supernatant was collected for detection of IL-2, IL-4, IL-5, IFN-gamma, and TNF by flow cytometry. RESULTS: ELISA showed that the mice in groups B, C, D, and E produced specific IgG and IgG1, IgG2a antibody isotypes, and the ratio of IgG2a/IgG1 was 1.73, 2.06, 2.44, and 3.09, respectively. The levels of IL-2, IFN-gamma and TNF in groups D and E were higher than that of groups B and C. The worm reduction rate and hepatic egg reduction rate in groups D (36.03%, 41.7%) and E (39.03%, 46.85%) were higher than those of groups B (26.28%, 28.35%) and C (28.38%, 31.39%) (P<0.01) . CONCLUSIONS: The codon optimized TPI DNA vaccine induces higher level of protective effect and Th1-biased cellular immune response than those of non-optimized TPI DNA vaccine.

Crystal Structure Analysis and Conformational Epitope Mutation of Triosephosphate Isomerase, a Mud Crab Allergen.

The triosephosphate isomerase (TIM), Scy p 8, is a crab allergen and shows cross-reactivity in the shellfish. Here, recombinant Scy p 8 was expressed, and its crystal structure was determined at a resolution of 1.8 Å. The three-dimensional structure of Scy p 8 is primarily composed of a (ß/α)8-barrel motif prototype. Additionally, Scy p 8 showed cross-reactivity with high sequential and secondary structural identity among TIMs from shellfish species. The site-directed mutagenesis of critical amino acids of conformational epitopes was carried out, and the mutants of Trp 168 and Lys 237 to Ala reduced immunoglobulin E (IgE)-binding activity by approximately 30%, compared with wild-type TIM in an inhibition ELISA; however, it still induced basophil activation despite the interpatient variability between patients. These results can help to provide an accurate template for the analysis of the IgE binding and establish meaningful relationships between structure and allergenicity.

Schistosoma mansoni triose phosphate isomerase peptide MAP4 is able to trigger naïve donor immune response towards a type-1 cytokine profile.

We evaluated the ability of naïve monocyte-derived dendritic cells (DC) to sensitize autologous peripheral blood mononuclear cells (PBMC) to the schistosome vaccine candidate MAP4 using a priming in vitro (PIV) assay. MAP4 is a multiple antigen peptide containing B- and T-cell epitopes derived from the glycolytic enzyme triose phosphate isomerase. PBMC primed and restimulated with MAP4 first and secondary recalls (MAP4 PIV cells) were examined for cell phenotype and cytokine production. We found that after the first recall stimulation with MAP4, the major cell population was predominantly CD4(+) T-cell subsets (68.5%), CD8(+high) (16%) and CD19(+) (10%). Additionally, MAP4 PIV cells significantly expressed CD4(+)-HLA-DR(+), -CD54(+), -CD45RO(+) (P < 0.0001) and -CD25(+) (P < 0.0004) together with significant expression of CD80(+) on CD19(+) B cells (P < 0.007). Cytokine production from activated MAP4 PIV cells was predominantly Th1-like, consisting mainly of IFN-gamma. Interestingly, IFN-gamma production was suppressed when Schistosoma mansoni-soluble egg antigen (SEA) was added to a MAP4 PIV cell culture. Furthermore, addition of MAP4 to a SEA PIV cell culture significantly reduced secretion of IL-10. The present findings add to the knowledge gained from studies in the mouse model, and our results show that naïve donor DC, sensitized with MAP4, were able to prime and clonally expand MAP4-specific T cells towards a Th1-type response.

Immunotherapeutic potential of Leishmania (Leishmania) donovani Th1 stimulatory proteins against experimental visceral leishmaniasis.

An effective therapeutic vaccination strategy is required for controlling visceral leishmaniasis (VL), a fatal systemic disease, through boosting the immunosuppressed state in Leishmania-infected individuals, as the majority of them living in the endemic regions exhibit either subclinical or asymptomatic infection which further often develops into a full-blown disease. Previously in our laboratory, several Th1 stimulatory recombinant proteins were successfully cloned, purified and assessed for their prophylactic efficacy against Leishmania challenge. Due to their immunostimulatory property, these proteins are needed to be evaluated for their immunotherapeutic potential in Leishmania-infected hamsters. Four proteins namely, aldolase, enolase, p45 and triose phosphate isomerase were taken up to immunize animals at different doses (50, 25 and 12.5 µg/animal). Immunization with lower doses of aldolase and enolase, i.e., 25 and 12.5 µg showed a significant decline (∼60%) in parasitic load along with an enhanced cellular immune response. These findings indicate that vaccination with above -stated Th1 stimulatory proteins is an effective immunotherapeutic approach against experimental VL. However, their efficacies may further be improved in combination with known therapeutic regimens or immunomodulators.

[Preliminary research on prokaryotic expression and immune protection of triosephosphate isomerase of Toxoplasma gondii].

OBJECTIVE: To study the prokaryotic expression and immune protection of triosephosphate isomerase (TPI) of Toxoplasma gondii in mice. METHODS: Total RNA was extracted from toxoplasma tachyzoites, and TPI fragment was amplified by PCR and cloned into the prokaryotic expression vector pET-28a (+). The target protein was induced with IPTG and purified by Ni-NTA affinity chromatography. The mice were immunized 4 times by emulsified TPI with adjuvant, and the last time was the strengthen immunization. At the same time, an adjuvant group and a normal group were set as controls. The blood samples were got from the tail vein of the mice, and the serum antibody titres were detected. All the mice were challenged with 400 toxoplasma tachyzoites to observe the survival time. RESULTS: The TPI gene was amplified from T. gondii cDNA by PCR. The recombinant vector TPI/pET-28a (+) was usefully constructed, and the TPI protein was expressed and purified. The serum antibody titre could be more than 100 thousand. After infected with toxoplasma tachyzoites, the survival time of the mice in the experimental group was longer than that of the mice in the control groups. CONCLUSIONS: The TPI protein of T. gondii could trigger the immunoprotection against T. gondii challenge in the mice.