Xiucai Xu

Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

Background Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission. Methodology/Principal Findings Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7). Conclusion/Significance A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.

Decrease of CD4+CD25+ regulatory T cells and TGF-β at early immune reconstitution is associated to the onset and severity of graft-versus-host disease following allogeneic haematogenesis stem cell transplantation

Graft-versus-host disease (GVHD) is a frequent and life threatening complication of allogeneic haematogenesis stem cell transplantation (aHSCT). The correlation of CD4(+)CD25(+) regulatory T cells (Tregs) in the patients after aHSCT to the occurrence and severity of acute and chronic GVHD (aGVHD and cGVHD) is not fully investigated. Here, we examined the levels of CD4(+)CD25(+) Tregs by assessment of CD4(+)CD25(high) and CD4(+)CD25(+)CD127(low) in peripheral blood, and the levels of serum TGF-beta and TNF-alpha in 56 patients at early immune reconstitution following aHSCT. Our data showed a significant reduction in the frequency of Tregs in patients with grades II-IV aGVHD and extensive cGVHD compared to healthy controls. Moreover, a decreased level of CD4(+)CD25(+) Tregs was correlated to increased severity of GVHD. The levels of CD4(+)CD25(+) Tregs in non-GVHD groups were however significantly higher than that in healthy controls. A significant decrease in the levels of TGF-beta and a significant increase the levels of TNF-alpha was also seen with increased severity of GVHD. This study suggested that measurement of CD4(+)CD25(+) Tregs along with serum TGF-beta and TNF-alpha at early immune reconstruction after aHSCT may indicate the onset and severity of both aGVHD and cGVHD.

Reactive Oxygen Species-Triggered Trophoblast Apoptosis Is Initiated by Endoplasmic Reticulum Stress via Activation of Caspase-12, CHOP, and the JNK Pathway in Toxoplasma gondii Infection in Mice

ABSTRACT Toxoplasma gondii infection in pregnant women may result in abortion or in fetal teratogenesis; however, the underlying mechanisms are still unclear. In this paper, based on a murine model, we showed that maternal infection with RH strain T. gondii tachyzoites induced elevated production of reactive oxygen species (ROS), local oxidative stress, and subsequent apoptosis of placental trophoblasts. PCR array analysis of 84 oxidative stress-related genes demonstrated that 27 genes were upregulated at least 2-fold and that 9 genes were downregulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The expression of NADPH oxidase 1 (Nox1) and glutathione peroxidase 6 (Gpx6) increased significantly, about 25-fold. The levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) increased significantly with T. gondii infection, and levels of glutathione (GSH) decreased rapidly. T. gondii infection increased the early expression of endoplasmic reticulum stress (ERS) markers, followed by cleavage of caspase-12, activation of ASK1/JNK, and increased apoptosis of trophoblasts, both in vivo and in vitro. The apoptosis of trophoblasts, the activation of caspase-12 and the ASK1/JNK pathway, and the production of peroxides were dramatically inhibited by pretreatment with N-acetylcysteine (NAC). The upregulation of Nox1 was contact dependent and preceded the increase in levels of ERS markers and the activation of the proapoptosis cascade. Thus, we concluded that apoptosis in placental trophoblasts was initiated predominantly by ROS-mediated ERS via activation of caspase-12, CHOP, and the JNK pathway in acute T. gondii infection. Elevated ROS production is the central event in T. gondii-induced apoptosis of placental trophoblasts.

The alteration of Th1/Th2/Th17/Treg paradigm in patients with type 2 diabetes mellitus: Relationship with diabetic nephropathy

T cells have been demonstrated to exert central roles in the development of type 2 DN (T2DN). To explore whether Th1/Th2/Th17/Treg paradigm plays an important role in the development of T2DN, we investigated the proportions of Th1/Th2/Th17/Treg cells and serum levels of relevant cytokines in T2DM patients with various degrees of nephropathy and controls. Moreover, we analyzed the relationships between the Th1/Th2/Th17/Treg paradigm or relevant cytokines with urine albumin:creatinine ratio (UACR). Our study demonstrated that the Th1/Th2/Th17/Treg paradigm skewed to Th1 and Th17 in T2DN patients. UACR was positively related to the proportions of Th1 and Th17 cells, as well as the ratio of Th17:Treg cells, and negatively related to the proportions of Treg cells. Furthermore, serum levels of IL-6, IL-17, IFN-γ, TNF-α, IL-2 and IL-10 were increased in T2DN patients, and positively related to UACR. These data indicate that the alteration of Th1/Th2/Th17/Treg paradigm exists in T2DN patients, which may contribute to the enhanced immune activation and inflammation, and subsequent development and progression of T2DN. These findings may provide one new approach to the underlying mechanisms of the development of T2DN.

Prevalence and genotypes of Toxoplasma gondii in pork from retail meat stores in Eastern China

Pork is known as one of the most important sources of Toxoplasma gondii infection in China. In the present study, 416 fresh pork samples were collected from different locations of Anhui province, Eastern China. Tissue fluid ELISA was conducted to detect the antibodies to T. gondii. Real-time PCR and bioassay were performed to identify the presence of T. gondii DNA and viable parasites, respectively. Seventy-five out of 416 samples (18.03%) demonstrated real-time PCR positive reaction and 42 out of 416 samples (10.1%) showed tissue fluid ELISA positive reaction. One isolate (Tgpkfx171) was obtained through bioassay in mice from 14 samples that demonstrated both PCR and ELISA positive reaction. The isolate and seven positive DNA samples were genotyped using 9 PCR-RFLP markers including SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Among these, only the isolate and two positive DNA samples were genotyped with complete data for all loci, belonging to ToxoDB#9 (Chinese 1) and ToxoDB#213, respectively. This is the first report of the prevalence and genetic typing of T. gondii from pork in retail meat stores in China. The present results provide an accurate picture of the risk of exposure to T. gondii in retail pork in China.

Toxoplasma gondii Virulence Factor ROP18 Inhibits the Host NF-κB Pathway by Promoting p65 Degradation

Background: ROP18 is a Toxoplasma secreted Ser/Thr protein kinase important for acute virulence. Results: ROP18 phosphorylates host p65 at Ser-468 and target this protein to the ubiquitin-dependent degradation. Conclusion: ROP18 inhibits the host NF-κB pathway by promoting p65 degradation. Significance: These findings reveal a novel molecular mechanism by which type I strain manipulates the host immune system to facilitate infection. The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remain unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser-468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12, and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.

Correlation of the CD4+CD25high T-regulatory cells in recipients and their corresponding donors to acute GVHD.

Graft‐versus‐host disease continues to be a major life‐threatening complication after allogeneic hematopoietic stem cells transplantation (aHSCT). The relationship of acute GVHD (aGVHD) with the levels of peripheral CD4+CD25high T cells in patients after aHSCT and in their corresponding donors is not fully investigated. We examined the levels of CD4+CD25high T cells in patients after aHSCT and in their corresponding donors, and analyzed the relationship of CD4+CD25high T cells to the incidence and prognosis of aGVHD. The recipients with normal or high CD4+CD25high T cells (three of eight, 37.5%) had no or mild aGVHD (grade I), and all survived during the follow‐up period. In striking contrast, the recipients with lower or no CD4+CD25high T cells suffered from greater than grade II aGVHD (four of four, 100%), and all died within 1‐year post‐aHSCT. Moreover, the number of CD4+CD25high T cells in recipients correlated significantly with that of their corresponding donors. The CD4+CD25high T cells from the recipients and their corresponding donors expressed high levels of Foxp3, and effectively suppressed the proliferation of CD4+CD25− responder T cells. This study suggests that human Treg cells may play an important role in aGVHD, as has been seen in murine models. The levels of peripheral CD4+CD25high T cells in recipients and donors could be helpful for predicting of the onset and outcome of aGVHD.

Changes of Treg-Associated Molecules on CD4+CD25+Treg Cells in Myasthenia Gravis and Effects of Immunosuppressants

ObjectiveMyasthenia gravis (MG) is a CD4+ T cell-dependent autoimmune disease, and close attention has been paid to the role of CD4+CD25+Treg cells (Tregs). Previous results regarding Tregs in MG patients have been conflicting. The discrepancy was partly ascribed to selecting different Treg-associated molecules in defining Tregs. Therefore, we considered it necessary to find a reliable index for assessing the immunologic state in MG patients and explore the effect of IS on them.MethodsWe adopted flow cytometric techniques to measure the numbers and frequencies of Tregs in peripheral blood taken from 57 patients and 91 age-matched healthy donors, and we also analyzed FOXP3 mean fluorescence intensity on Tregs.ResultsThe number and frequency of Tregs in peripheral blood of MG patients significantly decreased, together with down-regulation of FOXP3 expression. There was dynamic change of Treg cell level and the inverse relationship with clinical symptom, suggesting that the immunologic disorder in MG patients was related to peripheral Tregs population. Meanwhile, CD4+CD25+FOXP3+Helios+T cells might be activated Tregs, rather than nTregs. Moreover, the number and frequency of CD4+CD25+FOXP3+Helios+T cells significantly decreased in MG patients, indicating that the reduction of the activated Tregs population might be a critical contributor to the pathogenesis of MG.ConclusionsThe significant reduction of the peripheral Tregs population in MG patients might be responsible for the immunologic disorders in MG patients. IS such as GC took its effect possible by increasing the population size, and the underlying mechanism should be further investigated.

Trophoblast apoptosis through polarization of macrophages induced by Chinese Toxoplasma gondii isolates with different virulence in pregnant mice

Toxoplasma gondii is an apicomplexan parasite capable of transplacental transmission to cause spontaneous abortion or significant disease in the surviving neonate. Different from the dominant genotypes of T. gondii strains in European and North American which belong to three distinct clonal lineages, type I, type II, and type III, isolates from China possess the predominant genotype of China 1(ToxoDB#9) with a different virulence. The genotype-associated pathogenesis has been investigated previously. Based on two isolates of T. gondii from Chinese wild cats, a murine model of pregnancy and one transwell system in vitro, here we reported differentially polarized activation of macrophages induced by genotype China 1 strains, TgCtwh3 and TgCtwh6 with different virulence to mice, and its impact on trophoblast apoptosis. The results showed that macrophages were alternatively activated when infected with virulent TgCtwh3 while classically activated when infected with low virulent (cyst-forming) TgCtwh6 both in vitro and in vivo. By the analysis of flow cytometry, the percentage of the Th1 cells in two infection groups decreased significantly, and the Th2 cells from spleen escalated only in the virulent TgCtwh3 group. Interestingly, the high parasite burden was noted in the placenta of TgCtwh3-infected group whereas the inflammatory cells infiltration predominates in the TgCtwh6-infected group. In vivo trophoblast apoptosis in TgCtwh3 group was found to be more obvious when compared with TgCtwh6 although it was present in both. The present observations indicate that polarization of macrophages and modulation of Th subsets induced by the isolates with identical genotype but different virulence could contribute to trophoblast apoptosis through different mechanisms, suggesting a virulence-associated pathogenesis of T. gondii in abnormal pregnant outcome.

Toxoplasma gondii isolate with genotype Chinese 1 triggers trophoblast apoptosis through oxidative stress and mitochondrial dysfunction in mice

Congenital toxoplasmosis may result in abortion, severe mental retardation and neurologic damage in the offspring. Placental damage is considered as the key event in this disease. Here we show that maternal infection with Toxoplasma gondii Wh3 isolate of genotype Chinese 1, which is predominantly prevalent in China, induced trophoblast apoptosis of pregnant mouse. PCR array analysis of 84 key genes in the biogenesis and functions of mouse mitochondrion revealed that ten genes were up-regulated at least 2-fold in the Wh3 infection group, compared with those in the control. The elevated levels of reactive oxygen species (ROS), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG), as well as the decreased glutathione (GSH), were observed in the infected mice. The mRNA levels of NADPH oxidase 1 and glutathione peroxidase 6 (GPx6) were significantly increased. The production of excessive ROS was NADPH oxidase-dependent, which contributed to mitochondrial structural damage and mitochondrial dysfunction in placentas, followed by the cleavage of caspase-9 and caspase-3, and finally resulted in apoptosis of trophoblasts. All the above-mentioned phenomena were inhibited by pretreatment with the antioxidant of N-acetylcysteine (NAC). Taken together, we concluded that Wh3 infection during pregnancy may contribute to trophoblast apoptosis by oxidative stress-induced mitochondrial dysfunction and activation of the downstream signaling pathway.