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Objective To investigate the dynamic expression of cluster of differentiation 47 (CD47) and its ligands signaling regulatory protein α (SIRPα) and thrombospondin-1 (TSP-1) in mice infected with Toxoplasma gondii in the second and third trimesters.. Methods C57BL/6J mice (6 to 8 weeks old) were used for modeling T. gondii infection in the first trimester, and the pregnant mice were randomly divided into the normal control and infection groups, of 10 mice in each group. Pregnant mice in the infection group were intraperitoneally injected with 150 T. gondii tachyzoites on gestational day (Gd) 6.5, while pregnant mice in the normal control group were intraperitoneally injected with the same volume of physiological saline at the same time. The uterine and placental specimens were collected from all pregnant mice on Gd12.5 and Gd18.5, and the pregnant outcomes were recorded. The pathological damages of mouse uterine and placental specimens were observed using hematoxylin-eosin (HE) staining on Gd12.5 and Gd18.5. The relative expression of CD47, SIRPα, TSP-1, surface antigen 1 (SAG1), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-13 mRNA was quantified in mouse uterine and placental specimens using real-time fluorescence quantitative PCR (qPCR) assay, and the CD47, SIRPα, TSP-1 expression was determined in mouse uterine and placental specimens using immunohistochemical staining. Results As compared with those in the normal control group, the pregnant mice in the infection group showed back arching, bristling, trembling and listlessness during pregnancy, and several mice presented virginal bleeding and abortion. Pathological examinations showed inflammatory cell infiltration, congestion and necrosis in uterine and placental specimens of pregnant mice in the infection group, a higher abortion rate of pregnant mice was seen in the infection group than in the normal control group on Gd12.5 (χ2 = 20.405, P < 0.001) and Gd18.5 (χ2 = 28.644, P < 0.001). qPCR assay showed significant differences in the expression of CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 genes in mouse placental specimens between the normal control and infection groups on Gd12.5 and Gd18.5 [F′ (F) = 37.511, 29.337, 97.343, 53.755, 67.188, 21.145, 8.658 and 13.930, all P values < 0.001]. Higher CD47, SIRPα and TSP-1 gene expression was quantified in mouse placental specimens in the infection group than in the normal control group on Gd12.5 (all P values < 0.01), and lower CD47, SIRPα and TSP-1 gene expression was quantified in the infection group than in the normal control group on Gd18.5 (all P values < 0.001), while higher SAG1 gene expression was detected in placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01). In addition, higher INF-γ and IL-2 expression and lower IL-4 and IL-13 expression was detected in mouse placental specimens in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001), and there were significant differences in the CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 gene expression in uterine specimens of pregnant mice between the normal control and infection groups on Gd12.5 and Gd18.5 [H(F′ and F) = 14.951, 25.977, 18.711, 48.595, 39.318, 14.248 and 15.468, all P values < 0.01], and higher CD47 and TSP-1 expression was detected in mouse uterine specimens in the infection group than in the control group on Gd12.5 and Gd18.5 (all P values < 0.01); however, no significant difference was found in the SIRPα expression (P > 0.05). Higher SAG1 expression was detected in uterine specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01), and higher INF-γ and IL-2 gene expression and lower IL-4 and IL-13 gene expression was found in the placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001). Spearman correlation analysis showed that the CD47 gene expression correlated positively with IFN-γ (rs = 0.735, P < 0.05) and IL-2 (rs = 0.655, P < 0.05) and negatively with IL-4 (rs = −0.689, P < 0.05) and IL-13 expression (rs = −0.795, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd12.5, and the CD47 gene expression correlated negatively with IFN-γ (rs = −0.745, P < 0.05) and IL-2 expression (rs = −0.816, P < 0.05) and positively with IL-4 (rs = 0.704, P < 0.05) and IL-13 (rs = 0.802, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd18.5. Immunohistochemical staining showed mild CD47, SIRPα and TSP-1 expression in uterine and placental specimens of pregnant mice in the normal control group on Gd12.5 and Gd18.5, strong CD47, SIRPα and TSP-1 expression in the placental specimens of pregnant mice in the infection group on Gd12.5 and strong CD47 and TSP-1 expression in the uterine specimens of pregnant mice in the infection group on Gd12.5. Conclusions T. gondii infection in the first trimester may cause abnormal expression of CD47 and its ligands SIRPα and TSP-1 in the maternal-fetal interface of pregnant mice in the second and third trimesters, which may be associated with the immune escape of T. gondii at the maternal-fetal interface.
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Objective To predict the structure and antigenic epitope of the Strongyloides stercoralis serine protease inhibitor 1 (Ss-SRPN-1) protein using bioinformatics tools, and to construct prokaryotic expression plasmids for expression of recombinant Ss-SRPN-1 protein, so as to provide the basis for unraveling the function of the Ss-SRPN-1 protein. Methods The amino acid sequence of the Ss-SRPN-1 protein was downloaded from the NCBI database, and the physicochemical properties, structure and antigenic epitopes of the Ss-SRPN-1 protein were predicted using bioinformatics tools, including ExPASy, SWISS-MODEL and Protean. Primers were designed according to the nucleotide sequences of Ss-SRPN-1, and the Ss-SRPN-1 gene was amplified, cloned and sequenced with genomic DNA extracted from the infective third-stage larvae of S. stercoralis as a template. The Ss-SRPN-1 protein sequence was cloned into the pET28a (+) expression vector and transformed into Escherichia coli BL21 (DE) cells for induction of the recombinant Ss-SRPN-1 protein expression. The recombinant Ss-SRPN-1 protein was then purified and identified using Western blotting and mass spectrometry. Results Bioinformatics analysis showed that the Ss-SRPN-1 protein, which was composed of 372 amino acids and had a molecular formula of C1948H3046N488O575S16, was a stable hydrophilic protein, and the subcellular localization of the protein was predicted to be extracellular. The Ss-SRPN-1 protein was predicted to contain 11 dominant B-cell antigenic epitopes and 20 T-cell antigenic epitopes. The Ss-SRPN-1 gene with a length of 1 119 bp was successfully amplified, and the recombinant plasmid pET28a (+)/Ss-SRPN-1 was constructed and transformed into E. coli BL21(DE) cells. The expressed recombinant Ss-SRPN-1 protein had a molecular weight of approximately 43 kDa, and was characterized as a Ss-SRPN-1 protein. Conclusions The recombinant Ss-SRPN-1 protein has been expressed successfully, and this recombinant protein may be a potential vaccine candidate against strongyloidiasis.