PruΔcdpk2 Protects Pigs against Acute Toxoplasmosis Depending on T-Lymphocyte Subsets and Natural Killer Cell Responses.

Toxoplasma gondii is a widespread protozoan parasite approximately infecting one-third of the world population and can cause serious public health problems. In this study, we investigated the protective effect of the attenuated vaccine Pru:Δcdpk2 against acute toxoplasmosis and explored the underlying immune mechanisms of the protection in pigs. The systemic T-cell and natural killer (NK) cell responses were analyzed, including kinetics, phenotype, and multifunctionality (interferon [IFN]-γ, tumor necrosis factor [TNF]-α), and the IFN-γ levels were analyzed in PBMCs. Our results showed that T. gondii-specific antibodies were induced by Pru:Δcdpk2. After challenging with RH, the antibodies were able to respond quickly in the immunized group, and the expression level was significantly higher than that in the unimmunized group. The expression level of IFN-γ significantly increased after vaccination, and the CD3+ γδ-, NK, and CD3+ γδ+ cell subsets also significantly increased. At the same time, functional analysis indicated that these cells were polarized toward a Th1 phenotype, showing the ability to secrete IFN-γ and TNF-α. The CD4+CD8α-T cell population exhibited a higher frequency of IFN-γ+ producing cells compared with the CD4-CD8α+ and CD4+CD8α+ cell populations during the early days of vaccination. Our results indicated that the attenuated vaccine could induce the expression of NK, γδ, and CD3αß cells in pigs, and IFN-γ and TNF-α secreted by these cells are important for resistance to T. gondii infection.

Schistosoma japonicum cystatin alleviates paraquat poisoning caused acute lung injury in mice through activating regulatory macrophages.

BACKGROUND: Paraquat (PQ) is a widely used herbicide that poisons human by accident or intentional ingestion. PQ poisoning causes systemic inflammatory response syndrome (SIRS) resulting in acute lung injury (ALI) with an extremely high mortality rate. Blood trematode Schistosoma japonicum-produced cystatin (Sj-Cys) is a strong immunomodulatory protein that has been experimentally used to treat inflammation related diseases. In this study, Sj-Cys recombinant protein (rSj-Cys) was used to treat PQ-induced lung injury and the immunological mechanism underlying the therapeutic effect was investigated. METHODS: PQ-induced acute lung injury mouse model was established by intraperitoneally injection of 20 mg/kg of paraquat. The poisoned mice were treated with rSj-Cys and the survival rate was observed up to 7 days compared with the group without treatment. The pathological changes of PQ-induced lung injury were observed by examining the histochemical sections of affected lung tissue and the wet to dry ratio of lung as a parameter for inflammation and edema. The levels of the inflammation related cytokines IL-6 and TNF-α and regulatory cytokines IL-10 and TGF-ß were measured in sera and in affected lung tissue using ELISA and their mRNA levels in lung tissue using RT-PCR. The macrophages expressing iNOS were determined as M1 and those expressing Arg-1 as M2 macrophages. The effect of rSj-Cys on the transformation of inflammatory M1 to regulatory M2 macrophages was measured in affected lung tissue in vivo (EKISA and RT-PCR) and in MH-S cell line in vitro (flow cytometry). The expression levels of TLR2 and MyD88 in affected lung tissue were also measured to determine their role in the therapy of rSj-Cys on PQ-induced lung injury. RESULT: We identified that treatment with rSj-Cys significantly improved the survival rate of mice with PQ-induced lung injury from 30 % (untreated) to 80 %, reduced the pathological damage of poisoning lung tissue, associated with significantly reduced levels of proinflammatory cytokines (IL-6 from 1490 to 590 pg/ml, TNF-α from 260 to 150 pg/ml) and increased regulatory cytokines (IL-10 from360 to 550 pg/ml, and TGF-ß from 220 to 410 pg/ml) in both sera (proteins) and affected lung tissue (proteins and mRNAs). The polarization of macrophages from M1to M2 type was found to be involved in the therapeutic effect of rSj-Cys on the PQ-induced acute lung injury, possibly through inhibiting TLR2/MyD88 signaling pathway. CONCLUSIONS: Our study demonstrated the therapeutic effect of rSj-Cys on PQ poisoning caused acute lung injury by inducing M2 macrophage polarization through inhibiting TLR2/MyD88 signaling pathway. The finding in this study provides an alternative approach for the treatment of PQ poisoning and other inflammatory diseases.

Functional analyses of Toxoplasma gondii dihydroorotase reveal a promising anti-parasitic target.

Toxoplasma gondii relies heavily on the de novo pyrimidine biosynthesis pathway for fueling the high uridine-5'-monophosphate (UMP) demand during parasite growth. The third step of de novo pyrimidine biosynthesis is catalyzed by dihydroorotase (DHO), a metalloenzyme that catalyzes the reversible condensation of carbamoyl aspartate to dihydroorotate. Here, functional analyses of TgDHO reveal that tachyzoites lacking DHO are impaired in overall growth due to decreased levels of UMP, and the noticeably growth restriction could be partially rescued after supplementation with uracil or high concentrations of L-dihydroorotate in vitro. When pyrimidine salvage pathway is disrupted, both DHOH35A and DHOD284E mutant strains proliferated much slower than DHO-expressing parasites, suggesting an essential role of both TgDHO His35 and Asp284 residues in parasite growth. Additionally, DHO deletion causes the limitation of bradyzoite growth under the condition of uracil supplementation or uracil deprivation. During the infection in mice, the DHO-deficient parasites are avirulent, despite the generation of smaller tissue cysts. The results reveal that TgDHO contributes to parasite growth both in vitro and in vivo. The significantly differences between TgDHO and mammalian DHO reflect that DHO can be exploited to produce specific inhibitors targeting apicomplexan parasites. Moreover, potential DHO inhibitors exert beneficial effects on enzymatic activity of TgDHO and T. gondii growth in vitro. In conclusion, these data highlight the important role of TgDHO in parasite growth and reveal that it is a promising anti-parasitic target for future control of toxoplasmosis.

Mineralization Reduces the Toxicity and Improves Stability and Protective Immune Response Induced by Toxoplasma gondii.

Vaccination is an ideal strategy for the control and prevention of toxoplasmosis. However, the thermostability and effectiveness of vaccines limit their application. Here, calcium mineralization was used to fabricate Toxoplasma gondii tachyzoites as immunogenic core-shell particles with improved immune response and thermostability. In the current study, T. gondii RH particles coated with mineralized shells were fabricated by calcium mineralization. The mineralized shells could maintain the T. gondii tachyzoites structural integrity for at least 12 months and weaken the virulence. Immunization of mice with mineralized tachyzoites induced high levels of T. gondii-specific antibodies and cytokines. The immunized mice were protected with a 100% survival rate in acute and chronic infection, and brain cyst burdens were significantly reduced. This study reported for the first time the strategy of calcium mineralization on T. gondii and proved that mineralized tachyzoites could play an immune protective role, thus expanding the application of biomineralization in T. gondii vaccine delivery.