Toxoplasma gondii ROP17 inhibits the innate immune response of HEK293T cells to promote its survival.

Toxoplasma gondii secretes a group of rhoptry-secreted kinases (ROPs), which play significant roles in promoting intracellular infection. T. gondii rhoptry organelle protein 17 (ROP17) is one of these important effector proteins. However, its role in modulating host cell response during infection remains poorly understood. Here, we reveal that ROP17 (genotype I) induces significant changes in the expression genes and transcription factors of host cells. HEK293T cells were transfected with PCMV-N-HA-ROP17 plasmid or empty control PCMV-N-HA plasmid. Transcriptomic analysis revealed 3138 differentially expressed genes (DEGs) in PCMV-N-HA-ROP17-transfected HEK293T cells, including 1456 upregulated, 1682 downregulated DEGs. Also, 715 of the DEGs were transcription factors (TFs), including 423 downregulated TFs and 292 upregulated TFs. Most differentially expressed TFs, whether belong to signal transduction, cancer-related pathways or immune-related pathways, were downregulated in ROP17-expressing cells. ROP17 also decreased alternative splicing events in host cells, presumably via alteration of the expression of genes involved in the alternative splicing pathway. Taken together, our findings suggest a novel strategy whereby T. gondii ROP17 manipulates various cellular processes, including immune response through reprogramming host gene expression to promote its own colonization and survival in the infected host cells.

Immuno-efficacy of DNA vaccines encoding PLP1 and ROP18 against experimental Toxoplasma gondii infection in mice.

We constructed a new plasmid pIRESneo/ROP18/PLP1 that was injected intramuscularly into Kunming mice to evaluate its immune efficacy. The immunized mice exhibited significantly increased serum IgG2a levels, lymphocyte counts and Th1-type cytokine (IL-2, IL-12 and IFN-γ) levels. Moreover, the immunized mice exhibited longer survival times (44.7 ± 2.1 days for ROP18/PLP1 and 47.2 ± 2.9 days for ROP18/PLP1 + IL-18) and lower brain cyst burden (68.9% for ROP18/PLP1 and 72.4% for ROP18/PLP1 + IL-18) than control mice after T. gondii challenge. Our results demonstrate that the multiple-gene DNA vaccine including both ROP18 and PLP1 elicits greater protection against T. gondii challenge and stronger immunogenicity than single-gene vaccines.

Preparation of Open-Cell Long-Chain Branched Polypropylene Foams for Oil Absorption.

This study aimed to prepare open-cell foams using a blend of long-chain branched polypropylene and polyolefin elastomer (LCBPP/POE) for the production of reusable oil absorbents. The supercritical CO2 foaming process was conducted using a two-step batch rapid depressurization method. This unique two-step foaming approach significantly expanded the temperature and pressure windows, resulting in more uniform cells with smaller sizes, ultimately leading to higher expansion ratios and an increased open cell content. The foaming process was optimized by adjusting parameters, such as the LCBPP/POE ratio, foaming temperature, and foaming pressure, reaching a maximum open cell content of 97.6% and a maximum expansion ratio of 48. The influence of polypropylene (PP) crystallization was investigated with the aid of scanning electron microscopy and differential scanning calorimetry. Furthermore, the hydrophobic and lipophilic characteristics of the LCBPP/POE open-cell foam were determined via contact angle measurements and oil/water separation tests. Oil absorption tests revealed that the blended LCBPP/POE foam has a higher oil absorption capacity than that of the pure LCBPP foam. The cyclic oil absorption tests demonstrated the outstanding ductility and recoverability of the LCBPP/POE open-cell foam in comparison to those of the pure LCBPP foam. Over 10 cycles, the LCBPP/POE foam maintained a substantial adsorption capacity, retaining 99.3% of its initial oil absorption capacity. With its notable features, including a high open cell content, excellent hydrophobic and lipophilic characteristics, superior oil absorption capacity, impressive cyclic oil absorption performance, and robust reusability, LCBPP/POE open-cell foams exhibit significant promise as potential oil adsorbents for use in oil spill cleanup applications.

Toxoplasma gondii: protective immunity against experimental toxoplasmosis induced by a DNA vaccine encoding the perforin-like protein 1.

Toxoplasma gondii is an important zoonotic parasite infecting about one third of the world population, causing congenital infections and eye disease. T. gondii perforin-like protein 1 (TgPLP1) is believed to be involved in the acute virulence of T. gondii in mice, and is therefore of interest as a vaccine candidate. In this study, we constructed a DNA vaccine expressing TgPLP1, and evaluated the immune response in Kunming mice. The gene sequence encoding TgPLP1 was inserted into the eukaryotic expression vector pVAX I, and Kunming mice were immunized intramuscularly with the plasmid. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally with 1×10(3) tachyzoites of the virulent T. gondii RH strain. The results showed that pVAX/TgPLP1 alone or with pVAX/IL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/IL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization of pVAX/TgPLP1 with pVAX/IL-18 significantly (P<0.05) increased survival time (12.7±1.2days) of immunized mice, compared with pVAX/TgPLP1 alone (11.3±0.9days). These results demonstrate that TgPLP1 is a potential vaccine candidate against toxoplasmosis, worth further evaluation in other animal hosts. IL-18 could enhance the immune effect of TgPLP1, prolonging the survival time of immunized mice.

Evaluation of deoxynivalenol-induced toxic effects on DF-1 cells in vitro: cell-cycle arrest, oxidative stress, and apoptosis.

Deoxynivalenol (DON) is one of the most common mycotoxin contaminants of raw and processed cereal food. Lymphoid cells and fibroblasts are specified to be the most DON-sensitive cell types. In this study, we investigated the toxic effects of DON in chicken embryo fibroblast DF-1 cells. The results showed that DON significantly inhibited DF-1 cell viability in both a time- and concentration-dependent manner. DON could also inhibit the proliferation of DF-1 cells through G2/M phase arrest in the cell cycle progression. Moreover, oxidative stress induced by DON was indicated by increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), and decreased levels of glutathione (GSH) and superoxide dismutase (SOD). In addition, DON could also cause mitochondrial damage by decreasing the mitochondrial membrane potential and induce apoptosis accompanied with the up-regulation of apoptosis-related genes including Caspase-3, Caspase-8, Caspase-9, and AIFM1. These results suggested that DON could cause cell cycle arrest, oxidative stress, and apoptosis in DF-1 cells.

Gene expression profiling analysis of deoxynivalenol-induced inhibition of mouse thymic epithelial cell proliferation.

Deoxynivalenol (DON) is a mycotoxin produced as a secondary metabolite by fungal species. It has been shown that DON has serious toxic effects on many kinds of immune cells. However, the toxic effects on thymic epithelial cells were poorly understood. The purpose of this study is to investigate the gene expression differences for the DON-induced inhibition on the proliferation of mouse thymic epithelial cell line 1 (MTEC1). After the experiments of cell viability, morphological investigation and cell cycle analysis, microarray analysis was carried out. The differentially expressed genes belong to a variety of functional categories, including genes involved in metabolic process, cell cycle, oxidation-reduction process and apoptosis. Our results provide molecular insights into the gene expression differences of DON-induced toxic effects and suggest that p53 signaling pathway may play an important role in the inhibition of MTEC1 cell proliferation.

Vaccination with a DNA vaccine coding for perforin-like protein 1 and MIC6 induces significant protective immunity against Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is tightly related to microneme protein 6 (MIC6) and T. gondii perforin-like protein 1 (TgPLP1). In this study, we constructed a DNA vaccine expressing a TgPLP1/MIC6 fusion protein using the pIRESneo vector, and we evaluated the immune response induced by this vaccine in Kunming mice. Levels of IgG antibody, gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-12, IL-4, and IL-10 were examined. Five mice were chosen randomly from every group (vaccinated groups or the nonvaccinated control group) and were challenged intragastrically with 80 cysts of T. gondii strain PRU (genotype II) in order to observe mortality daily. To analyze protection against a less-virulent challenge, eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. The brain parasite load was evaluated 6 weeks after infection. The results demonstrated that immunization with pIRESneo/MIC6/PLP1 resulted in the lowest brain cyst count and prolonged the survival time of immunized mice. The levels of Toxoplasma-specific IgG, IFN-γ, IL-2, and IL-12 increased significantly, and the numbers of cysts in brains decreased more obviously, in the group immunized with plasmid pIRESneo/MIC6/PLP1 than in the other groups (P < 0.05). Compared with pIRESneo/MIC6/PLP1, coimmunization with pIRESneo/MIC6/PLP1 and adjuvant murine IL-18 promoted cellular and humoral immune responses but did not contribute significantly to cyst reduction (65.43% versus 61.60%) or the survival of immunized mice (45.0 ± 2.9 days versus 42.8 ± 2.9 days) (P > 0.05). Furthermore, the study also showed that the immune efficacy induced by pIRESneo/MIC6/PLP1 was better than that induced by pVAX/PLP1 or pVAX/MIC6 alone.

Genetic characterization of Toxoplasma gondii isolates from pigs in southwestern China.

The genetic diversity of Toxoplasma gondii varies in different geographical regions. Isolates of T. gondii in South America, for example, are genetically and biologically divergent from those in North America and Europe, where the population structure is highly clonal and composed mainly of 3 distinct lineages, i.e., Types I, II, and III. However, little is known of the T. gondii genotypes in the People's Republic of China. Toxoplasma gondii infection in pigs causes significant economic loss and presents a risk for human infection. We conducted a survey to determine the genetic diversity of this parasite in slaughtered pigs from Yunnan Province, southwestern China. In total, 412 DNA samples were extracted from hilar lymph nodes and livers of pigs from slaughterhouses in Yunnan Province in southwest China, 56 of which were found to be positive for the T. gondii SAG3 gene. These positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 5 isolates were genotyped with complete data for all loci. Only 1 genotype (ToxoDB 9) was identified, previously reported as a widespread lineage from pigs, cats, and human patients in China. The results indicate that this genotype may be the major T. gondii lineage in China and possibly all of eastern Asia. This is the first report of genetic typing of T. gondii isolates from pigs in China's southwestern Yunnan Province, the results of which have implications for the prevention and control of T. gondii infections in humans and other animals.