Yuanhong Xu

Genotyping of Toxoplasma gondii isolates from cats in different geographic regions of China.

Fourteen isolates of Toxoplasma gondii were isolated from cats from 4 different geographic provinces (Anhui, Hubei, Shanxi and Guangdong) in China and their genetic diversity with 8 nuclear loci SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico, was analysed by restriction fragment length polymorphisms (RFLPs). Two genotypes from these 14 isolates were identified but none of them belongs to the typical genetic types (types I, II and III). It is unexpected that such high similarity was observed in these 14 isolates although their original regions are significantly distant. Our results strongly indicate that the three traditional clonal lineages of types I, II and III of this parasite may not be preponderant in China. In addition, our results show that the genotypes of T. gondii in China may be highly clonal with atypical genotypes and higher virulence.

Impact of Schistosoma japonicum Infection on Collagen-Induced Arthritis in DBA/1 Mice: A Murine Model of Human Rheumatoid Arthritis

Background The hygiene hypothesis suggests that helminth infections prevent a range of autoimmune diseases. Methodology/Principal Findings To investigate the effects of S. japonicum infection on collagen-induced arthritis (CIA), male DBA/1 mice were challenged with unisexual or bisexual S. japonicum cercariae two weeks prior to bovine type II collagen (CII) immunization or at the onset of CIA. S. japonicum infection prior to CII immunization significantly reduced the severity of CIA. ELISA (enzyme linked immunosorbent assay) showed that the levels of anti-CII IgG and IgG2a were reduced in prior schistosome-infected mice, while anti-CII IgG1 was elevated. Splenocyte proliferation against both polyclonal and antigen-specific stimuli was reduced by prior schistosome infection as measured by tritiated thymidine incorporation (3H-TdR). Cytokine profiles and CD4+ T cells subpopulation analysis by ELISA and flow cytometry (FCM) demonstrated that prior schistosome infection resulted in a significant down-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β and IL-6) and Th1 cells, together with up-regulation of the anti-inflammatory cytokine IL-10 and Th2 cells. Interestingly, the expansion of Treg cells and the reduction of Th17 cells were only observed in bisexually infected mice. In addition, prior schistosome infection notably reduced the expression of pro-inflammatory cytokines and receptor activator of NF-κB ligand (RANKL) in the inflamed joint. However, the disease was exacerbated at one week after infection when established CIA mice were challenged with bisexual cercariae. Conclusion/Significance Our data provide direct evidence that the Th2 response evoked by prior S. japonicum infection can suppress the Th1 response and pro-inflammatory mediator and that bisexual infection with egg-laying up-regulates the Treg response and down-regulates the Th17 response, resulting in an amelioration of autoimmune arthritis. The beneficial effects might depend on the establishment of a Th2-dominant response rather than the presence of the eggs. Our results suggest that anti-inflammatory molecules from the parasite could treat autoimmune diseases.

Toxoplasma gondii prevalence in food animals and rodents in different regions of China: isolation, genotyping and mouse pathogenicity

BackgroundRecent studies of Toxoplasma gondii isolates from animals in different regions of China have shown a limited genetic diversity and type China 1 was the dominant genotype of T. gondii prevalent in Chinese animals. However, little has been known concerning the isolation and genotyping of T. gondii circulating in chickens, pigs and rodents in China. The aim of the study was to characterize samples of T. gondii isolates obtained from naturally infected cats, pigs and free-range chickens slaughtered for human consumption in China.MethodsIn the present study, brain tissues of 77 animals collected from different areas of China, including 24 free-range chickens (Gallus domesticus) , 13 voles (Rattus flavipectus), 23 pigs and 17 cats, were bioassayed in mice and viable T. gondii were isolated from the brains of eleven. These eleven T. gondii isolates were maintained in Kunming (KM) outbred mice and DNA isolated from tissues of infected mice was characterized using 11 PCR-restriction fragment length polymorphism (PCR-RFLP) markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. Moreover, to determine mouse virulence of China 1 lineage of parasites, a TgCtgy5 genotype isolate was selected randomly and assessed in KM mice with different inoculation doses.ResultsResults of genotyping revealed that ten isolates were type China 1 (ToxoDB PCR-RFLP genotype #9), and TgCksz1 was a new genotype that was reported for the first time designated here as ToxoDB PCR-RFLP #225. No clonal types I, II and III lineages were found. DNA sequencing of four introns (EF1, HP2, UPRT1 and UPRT7) and two genes (GRA6 and GRA7) from representative isolates confirmed the results of PCR-RFLP genotyping. The TgCtgy5 isolate was highly virulent in KM mice; all infected mice died of acute toxoplasmosis, irrespective of the inoculation dose. The results indicate that mouse virulent isolates of T. gondii are predominantly circulating in cats in China.ConclusionsT. gondii isolated from chickens, pigs, cats and rodents in different locations in China were genotyped and the results reconfirmed the limited diversity of T. gondii in China and showed that type China 1 lineage was dominant in this country.

Variation detection based on next-generation sequencing of type Chinese 1 strains of Toxoplasma gondii with different virulence from China.

BackgroundToxoplasma gondii is an intracellular protozoan that affects most species of endothermic animals including humans with a great infection rate. The vertical transmission of T. gondii causes abortion, constituting a serious threat to humans and leading to great losses in livestock production. Distinct from population structure of T. gondii in North America and Europe, Chinese 1 (ToxoDB #9) is a dominant genotype prevalent in China. Among the isolates of Chinese 1, the Wh3 and Wh6 have different virulence and pathogenicity in mice. However, little has been known about their difference at the genomic level. Thus the next-generation sequencing (NGS) approach was used to discover the association of the phenotypical variations with the genome sequencing data and the expression and polymorphisms of the key effectors.ResultsWe successfully sequenced the genome of Chinese 1 strains of Wh3 and Wh6. The average sequencing depths were 63.91 and 63.61 for Wh3 and Wh6, respectively. The variations of both isolates were identified in comparison with reference genome of type I GT1 strain. There were 505,645 and 505,856 SNPs, 30,658 and 30,004 indels, 4661 and 2320 SVs, and 1942 and 3080 CNVs for Wh3 and Wh6, respectively. In target search variations of particular factors of T. gondii, the dense granule protein 3 (GRA3) and rhoptry neck protein 3 (RON3) were found to have 35 SNPs, 2 indels and 89 SNPs, 6 indels, respectively. GRA3 and RON3 were both found to have higher expression levels in less virulent Wh6 than in virulent Wh3. Both strains of type Chinese 1 share polymorphic GRA15II and ROPI/III with type I, II, and III strains.ConclusionsSequencing of the two strains revealed that genome structure of Chinese 1 and type I strains has considerable genomic variations. Sequencing and qRT-PCR analyses of 26 effectors displayed a remarkable variation that may be associated with phenotype and pathogenic differences.

Activated microglia contribute to neuronal apoptosis in Toxoplasmic encephalitis

BackgroundA plethora of evidence shows that activated microglia play a critical role in the pathogenesis of the central nervous system (CNS). Toxoplasmic encephalitis (TE) frequently occurs in HIV/AIDS patients. However, knowledge remains limited on the contributions of activated microglia to the pathogenesis of TE.MethodsA murine model of reactivated encephalitis was generated in a latent infection with Toxoplasma gondii induced by cyclophosphamide. The neuronal apoptosis in the CNS and the profile of pro-inflammatory cytokines were assayed in both in vitro and in vivo experiments.ResultsMicroglial cells were found to be activated in the cortex and hippocampus in the brain tissues of mice. The in vivo expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) were up-regulated in TE mice, and accordingly, the neuronal apoptosis was significantly increased. The results were positively correlated with those of the in vitro experiments. Additionally,apoptosis of the mouse neuroblastoma type Neuro2a (N2a) remarkably increased when the N2a was co-cultured in transwell with microglial cells and Toxoplasma tachyzoites. Both in vivo and in vitro experiments showed that minocycline (a microglia inhibitor) treatment notably reduced microglial activation and neuronal apoptosis.ConclusionsActivated microglia contribute to neuronal apoptosis in TE and inhibition of microglia activation might represent a novel therapeutic strategy of TE.

miR-429 functions as a tumor suppressor by targeting FSCN1 in gastric cancer cells

It has been previously reported that the deregulation of microRNAs in gastric cancer (GC) was correlated with the progression and prognosis. miR-429, a member of the miR-200 family, was previously shown to play an important role in human carcinomas. Our study shows that miR-429 is significantly downregulated in GC tissues compared with matched nontumor tissues. Overexpression of miR-429 in GC cells suppressed cell proliferation. Fascin-1 (FSCN1) was identified as one of the targets of miR-429 and knockdown of FSCN1 mimics the function of miR-429 overexpression. In conclusion, miR-429 acts as a tumor suppressor by targeting FSCN1, suggesting that miR-429 and FSCN1 can both be potential therapeutic targets of GC.

Tim-2 up-regulation and galectin-9-Tim-3 pathway activation in Th2-biased response in Schistosoma japonicum infection in mice.

T cell immunoglobulin domain and mucin domain (Tim) family, a new gene that expresses on the surface of T cells, plays a critical role in regulation of T cells response. Previous data have shown that Tim-3 expressed on Th1 cells promotes itself apoptosis. Tim-2 is preferentially up-regulated during Th2 differentiation and functions as a potent costimulatory molecule for T-cell immunity. The present study aims to learn whether Tims are responsible for Th2-biased response evoked by Schistosoma japonicum infection. The expressions of Tim-2 and Tim-3 in spleen lymphocytes from S. japonicum-infected mice were examined, and the possible role of galectin-9-Tim-3 pathway in Th2-biased response triggered by schistosome infection was discussed. Our results showed that Tim-2 mRNAs were up-regulated in the spleen of schistosome-infected mice, which coincided with elevated IL-4 gene expression. Administration of galectin-9 significantly induced apoptosis of naïve spleen lymphocytes with down-regulation IFN-γexpression in vitro. Additionally, Tim-3-Fc fusion protein notably enhanced Th1 cells and decreased Th2 cells in vitro. Thus, we concluded that pro-apoptotic effects on Th1 population through galectin-9-Tim-3 pathway and the up-regulation of Tim-2 on Th2 cells might be critical to Th2-biased response of host with schistosomiasis japonica.

Toxoplasma gondii GRA15II effector-induced M1 cells ameliorate liver fibrosis in mice infected with Schistosomiasis japonica.

Recent studies indicated that type II Toxoplasma gondii (Tg) GRA15II favored the generation of classically activated macrophages (M1), whereas type I/III TgROP16I/III promoted the polarization of alternatively activated macrophages (M2). A number of studies have demonstrated that M2 cells are involved in the pathogenesis of the liver fibrogenesis caused by Schistosoma japonicum. The purpose of the present study was to explore the inhibitory effect of Toxoplasma-derived TgGRA15II on mouse hepatic fibrosis with schistosomiasis. The gra15II and rop16I/III genes were amplified from strains T. gondii PRU and Chinese 1 Wh3, respectively. Lentiviral vectors containing the gra15II or rop16I/III plasmid were constructed and used to infect the RAW264.7 cell line. The polarization of the transfected cells was evaluated, followed by co-culture of the biased macrophages with mouse hepatic stellate JS1 cells. Then, mice were injected with GRA15II-driven macrophages via the tail vein and infected with S. japonicum cercariae. TgGRA15II induced a M1-biased response, whereas TgROP16I/III drove the macrophages to a M2-like phenotype. The in vitro experiments indicated that JS1 cell proliferation and collagen synthesis were decreased following co-culture with TgGRA15II-activated macrophages. Furthermore, mice inoculated with TgGRA15II-biased macrophages displayed a notable alleviation of collagen deposition and granuloma formation in their liver tissues. Our results suggest that TgGRA15II-induced M1 cells may dampen the M2 dominant pathogenesis of hepatic fibrosis and granulomatosis. These results provide insights into the use of parasite-derived immunomodulators as potential anti-fibrosis agents and to re-balance the schistosomiasis-induced immune response.

Schistosoma japonicum cystatin attenuates murine collagen-induced arthritis

Recombinant SjCystatin (rSjCystatin), a recombinant protein of Schistosoma japonicum cystatin, has been reported to have an effect on immunoregulation mediated by IL-10 induction. Rheumatoid arthritis (RA) is a common autoimmune inflammatory arthropathy, and recombinant immune-modulating drugs for RA treatment are under development. We aimed to study the putative immune regulation of rSjCystatin and its prophylactic/therapeutic effects on murine collagen-induced arthritis (CIA). CIA was induced in DBA/1 mice by inoculation with bovine collagen II (CII). rSjCystatin was administered prior or post development of CIA. The severity of CIA was assessed using established clinical and histopathological scoring systems. The incidence was also determined. The CII-specific antibodies in sera and cytokines in splenocyte culture supernatants were measured by ELISA. Th1/Th2/Th17 cells and Tregs development in splenocytes were monitored by flow cytometry. The inflammatory mediators in the diseased joint were semiquantitated by qPCR. Prophylactic injection of rSjCystatin attenuated paw clinical scores, incidence, and histopathology scores of joints in CIA mice. The arthritis-alleviative effects were closely associated with the augmentation of IL-4, IL-10, and collagen-specific IgG1, and with the distinct reduction of IFN-γ, collagen-specific IgG2a, and the marked decrease of proinflammatory cytokines IL-6, IL-17, and TNF-α and RANKL. The data indicate that rSjCystatin may prevent cartilage destruction and inflammation of joints in CIA mice. The effects are related to the inhibitory modulation of Th1 and Th17 and upregulation of Tregs and Th2 via a shift of cytokines profiling from Th1 to Th2 response.

Prevalence of Toxoplasma gondii infection in HIV-infected patients and food animals and direct genotyping of T. gondii isolates, Southern Ghana

Toxoplasma gondii is of public health and veterinary importance causing severe diseases in immunocompromised individuals including HIV/AIDS patients and in congenital cases and animals. There is limited information on the epidemiology of T. gondii infection in humans, particularly HIV patients and food animals and the parasite genotypes in Ghana. A total of 394 HIV-infected patients from three hospitals were screened for T. gondii anti-IgG and IgM using ELISA. DNAs from blood samples of seropositve participants and 95 brain tissues of food animals were PCR assayed to detect Toxoplasma gra6. DNA positive samples were genotyped using multilocus nested polymerase chain reaction restriction fragment length polymorphism at 10 loci: sag1, alt.sag2, sag3, btub, gra6, l358, c22-8, c29-2, pk1, and apico. The overall seroprevalence was 74.37% (293/394). Toxoplasma DNAs were detected in 3.07% of the seropositive participants and 9.47% of the animals. Six of the human DNA positive samples were partly typed at sag3: 33.33, 50, and 16.67% isolates had type I, II, and III alleles, respectively. All nine isolates from food animals typed at nine loci except apico were atypical: six isolates were identical to ToxoDB #41 and #145, and one was identical to TgCkBrRj2 all identified in Brazil. The genotype of two isolates has not been reported previously and was named as TgCtGh1. T. gondii seroprevalence is high among the HIV-infected individuals with T. gondii circulating in Ghana being genetically diverse.