Production and characterization of monoclonal antibodies against Toxoplasma gondii ROP18 with strain-specific reactivity.

The rhoptry kinase 18 of Toxoplasma gondii (TgROP18) has been identified as a key virulence factor that allows the parasite to escape from host immune defences and promotes its proliferation in host cells. Although much research is focused on the interaction between host cells and TgROP18, the development of monoclonal antibodies (mAbs) against TgROP18 has not been reported till date. To produce mAbs targeting TgROP18, two hybridomas secreting mAbs against TgROP18, designated as A1 and T2, were generated using cell fusion technology. The subtypes of the A1 and T2 mAbs were identified as IgG3 λ and IgM κ, and peptide scanning revealed that the core sequences of the antigenic epitopes were 180LRAQRRRSELVFE192 and 351NYFLLMMRAEADM363, respectively. The T2 mAb specifically reacted with both T. gondii type I and Chinese I, but not with T. gondii type II, Plasmodium falciparum or Schistosoma japonicum. Finally, the sequences of heavy chain and light chain complementarity-determining regions of T2 were amplified, cloned and characterized, making the modification of the mAb feasible in the future. The development of mAbs against TgROP18 would aid the investigation of the molecular mechanisms underlying the modulation of host cellular functions by TgROP18, and in the development of strategies to diagnose and treat Toxoplasmosis.

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.

Toxoplasma gondii virulence factor ROP18 inhibits the host NF-κB pathway by promoting p65 degradation.

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.

The effect of recombinant sTGFß1RII and sIL13Rα2 receptor proteins on schistosomiasis japonica, hepatic fibrosis and signal transduction in a mouse model of schistosome disease.

This study was designed to investigate the effect of recombinant sTGFß1RII and sIL13Rα2 receptor proteins on schistosomiasis japonica, hepatic fibrosis and the expression of SMAD3 and STAT6. The proteins sTGFß1RII and sIL13Rα2 were expressed in Escherichiacoli, purified using affinity chromatography and characterized by Western blotting. Female BALB/C mice (48) were randomly divided into eight groups and infected with Schistosoma japonicum. Five weeks after infection, test groups were injected with the recombinant proteins at different doses. Eight weeks after infection, lung and hepatic tissue samples were obtained and stained with hematoxylin and eosin (HE) and Masson's trichrome. Immunohistochemical staining was used to detect the expression of SMAD3 and STAT6. The recombinant proteins sTGFß1RII and sIL13Rα2 were successfully expressed, purified, and characterized. The granuloma area, hepatic hydroxyproline (HYP) level and hepatic fibrosis of the protein therapeutic groups were significantly smaller than those of the positive control group (P<0.01). Treatment with sTGFß1RII was more effective when the protein was administered for 4weeks rather than 2 (P<0.01). Hepatic fibrosis in the groups using a low dose of protein sTGFß1 was lower that of the combination group (P<0.05). The expression level of STAT6 was significantly lower in groups treated with sIL13Rα2 than in groups not treated with the protein (P<0.01). The recombinant proteins TGFß1RII and sIL13Rα2 were able to decrease granuloma area and hepatic fibrosis in schistosomiasis japonica, and also reduced the expression of the signal transduction proteins SMAD3 and STAT6. The proteins were more effective when used in combination than when applied singly.

Knockdown of DJ-1 Exacerbates Neuron Apoptosis Induced by TgCtwh3 through the NF-κB Pathway.

Mutations or loss of function of DJ-1 and Toxoplasma gondii (T. gondii) infection has been linked to neurodegenerative diseases, which are often caused by oxidative stress. However, the relationship between DJ-1 and T. gondii infection is not yet fully understood. Therefore, this study aimed to investigate the expression of DJ-1 in the hippocampus tissue of mice or in HT22 infected with T. gondii Chinese 1 genotype Wh3 strain (TgCtwh3) and the effect of DJ-1 knockdown on neuronal apoptosis induced by TgCtwh3 tachyzoite, as well as the underlying mechanism at the cellular and molecular level. Firstly, we detected DJ-1 protein expression and cell apoptosis in the hippocampal tissue of mice infected by TgCtwh3. Then, we examined DJ-1 expression and apoptosis in HT22 challenged with TgCtwh3. Finally, we evaluated the apoptosis in HT22 with DJ-1 knockdown which was infected with TgCtwh3 and assayed the expression of NF-κBp65 and p-NF-κBp65. Our results showed that DJ-1 expression was reduced and neurons underwent apoptosis in the hippocampus of mice infected with TgCtwh3 tachyzoites. Additionally, the knockdown of DJ-1 followed by infection with TgCtwh3 tachyzoites led to increased apoptosis in HT22 cells through the NF-κB signaling pathway. Therefore, this study suggests that DJ-1 is an important target for preventing apoptosis caused by T. gondii TgCtwh3.

Studies on the mechanism of Toxoplasma gondii Chinese 1 genotype Wh6 strain causing mice abnormal cognitive behavior.

BACKGROUND: Alzheimer's disease presents an abnormal cognitive behavior. TgCtwh6 is one of the predominant T. gondii strains prevalent in China. Although T. gondii type II strain infection can cause host cognitive behavioral abnormalities, we do not know whether TgCtwh6 could also cause host cognitive behavioral changes. So, in this study, we will focus on the effect of TgCtwh6 on mouse cognitive behavior and try in vivo and in vitro to explore the underlying mechanism by which TgCtwh6 give rise to mice cognitive behavior changes at the cellular and molecular level. METHODS: C57BL/6 mice were infected orally with TgCtwh6 cysts. From day 90 post-infection on, all mice were conducted through the open field test and then Morris water maze test to evaluate cognitive behavior. The morphology and number of cells in hippocampus were examined with hematoxylin-eosin (H&E) and Nissl staining; moreover, Aß protein in hippocampus was determined with immunohistochemistry and thioflavin S plaque staining. Synaptotagmin 1, apoptosis-related proteins, BACE1 and APP proteins and genes from hippocampus were assessed by western blotting or qRT-PCR. Hippocampal neuronal cell line or mouse microglial cell line was challenged with TgCtwh6 tachyzoites and then separately cultured in a well or co-cultured in a transwell device. The target proteins and genes were analyzed by immunofluorescence staining, western blotting and qRT-PCR. In addition, mouse microglial cell line polarization state and hippocampal neuronal cell line apoptosis were estimated using flow cytometry assay. RESULTS: The OFT and MWMT indicated that infected mice had cognitive behavioral impairments. The hippocampal tissue assay showed abnormal neuron morphology and a decreased number in infected mice. Moreover, pro-apoptotic proteins, as well as BACE1, APP and Aß proteins, increased in the infected mouse hippocampus. The experiments in vitro showed that pro-apoptotic proteins and p-NF-κBp65, NF-κBp65, BACE1, APP and Aß proteins or genes were significantly increased in the infected HT22. In addition, CD80, pro-inflammatory factors, notch, hes1 proteins and genes were enhanced in the infected BV2. Interestingly, not only the APP and pro-apoptotic proteins in HT22, but also the apoptosis rate of HT22 increased after the infected BV2 were co-cultured with the HT22 in a transwell device. CONCLUSIONS: Neuron apoptosis, Aß deposition and neuroinflammatory response involved with microglia polarization are the molecular and cellular mechanisms by which TgCtwh6 causes mouse cognitive behavioral abnormalities.

Paeoniflorin attenuates schistosomiasis japonica-associated liver fibrosis through inhibiting alternative activation of macrophages.

Interleukin (IL)-13 and alternatively activated macrophages (AAMs) play an important role in liver granuloma and fibrosis of schistosomiasis. Paeoniflorin (PAE, C23H28O11) has been reported to have an anti-hepatic fibrosis effect in schistosomiasis; however, the mechanism has not been fully elucidated. In this study, we measured serum hyaluronic acid (HA) concentrations, liver granuloma diameter and volume density, fibrosis degree and expressions of IL-13, arginase-1 (ARG-1), nitric oxide synthase-2 (NOS-2), and phosphorylated signal transducer and activator of transcription 6 (p-STAT6) in mice liver of schistosomiasis. Then we detected expressions of specific biomarkers of AAMs and activity of Arg-1 in Kupffer cells (KCs) from infected and PAE-treated mice, or in KCs from uninfected mice, but exposed to rIL-13 in vitro. Finally, we observed expression of IL-13 signalling molecules in KCs and secretion of IL-13 from lymphocytes of infected and PAE-treated mice. Our results showed that during schistosomiasis, IL-13 expression and secretion increased with liver macrophages activated alternatively. PAE not only directly inhibited alternative activation of macrophages via reducing the phosphorylations of janus-activated kinase 2 (JAK2) and/or STAT6, leading to reduction of AAMs-related markers and Arg-1 activity, but also indirectly suppressed alternative activation of macrophages through decreasing secretion of IL-13. PAE might be a promising prophylactic agent for hepatic granuloma and fibrosis of schistosomiasis japonica.

[Inhibitory effect of paeoniflorin on the collagen production by fibroblasts through IL-13/STAT6 signaling pathway].

OBJECTIVE: To observe the effects of paeoniflorin on 3T3 fibroblast activation, proliferation and collagen production through IL-13/STAT6 signaling pathway. METHODS: 3T3 cell strain was cultured with serum-free medium for 12 h, then stimulated by paeoniflorin (200, 400, 600, 800, and 1000 mg/L) or rIL-13 (6.25, 12.5, 50, 100, and 200 microg/L) for another 24 h. At the same time the blank control group for paeoniflorin or rIL-13 was observed. 3T3 cell proliferation was assayed by Cell Counting Kit-8 (CCK-8), and an appropriate concentration (100 microg/L) of rIL-13 was chosen according to the result of cell proliferation. Subsequently, 3T3 cell cultured with serum-free medium for 12 h was stimulated by 100 microg/L rIL-13 for 12 h, and then was treated with different concentrations of paeoniflorin (200, 400, 600, 800, and 1000 mg/L) for another 24 h. Untreated 3T3 cell served as blank control Cell proliferation was measured by CCK-8. Hydroxyproline content in cell supernatant was determined by alkaline lysis method. IL-13Ralpha1, alpha-SMA and STAT6 protein expression were detected by Western blotting. Col-I, Col-III, IL-13Ralpha1 and STAT6 mRNA expression were analyzed by RT-PCR. RESULTS: Paeoniflorin inhibited 3T3 cell proliferation in a concentration-dependent manner (r = -0.980, P < 0.01), and there was a statistically significant difference among all groups (F = 198.599, P < 0.01). rIL-13 caused a remarkably concentration-dependent increase in proliferation of 3T3 cells (r = 0.538, P < 0.05). Paeoniflorin (200, 400, 600, 800, and 1000 mg/L) inhibited proliferation of 3T3 cell stimulated by rIL-13 in a concentration-dependent manner (1.780 +/- 0.177, 1.636 +/- 0.073, 0.965 +/- 0.066, 0.623 +/- 0.037, 0337 +/- 0.022, r = -0.971, P < 0.01), and among all groups there existed a significant difference (F = 198.537, P < 0.01). Moreover, paeoniflorin also suppressed secretion of hydroxyproline from 3T3 cell stimulated by rIL-13 in a concentration-dependent manner (3.030 +/- 0.094, 2.976 +/- 0.047, 2.814 +/- 0.047, 2.652 +/- 0.124, 2.408 +/- 0.124, r = -0.916, P < 0.01) with a statistical significance among all groups (F = 13.642, P < 0.01). Further investigations showed that paeoniflorin decreased both protein expression of alpha-SMA, IL-13Ralpha1, and STAT6, and mRNA expression of Col-I, Col-III, IL-13Ralpha1, and STAT6 in 3T3 cell stimulated by rIL-13. CONCLUSION: Paeoniflorin inhibits activation, proliferation of fibroblasts and production of collagen from fibroblasts through IL-13/STAT6 signaling pathway, which might be one of mechanisms of anti-hepatic fibrosis of paeoniflorin in schistosomiasis japonica.

Evaluation of recombinant SjLAP and SjFBPA in detecting antibodies to Schistosoma japonicum.

OBJECTIVE: To investigate the early response of immunoglobulin G (IgG) antibody responses to Schistosoma japonicum infection in mice by using the recombinant proteins, S. japonicum leucine aminopeptidase (rSjLAP) and S. japonicum fructose-1, 6-bisphosphate aldolase (rSjFBPA), and evaluate the potential of rSjLAP and rSjFBPA in diagnosis as well as in assessment of therapeutic efficacy in human schistosomiasis. METHODS: rSjLAP or rSjFBPA was induced from Escherichia coli BL21 strain transfected with the expression vectors, pET-28a-rSjFBPA/BL21 or pET-28a-rSjLAP/BL21 using isopropyl-beta-D-thiogalactoside (IPTG), and purified by Ni-NTA His Bind resin. 88 BALB/c female mice, inbred and 6 to 8 weeks old, were randomly divided into 4 groups. Groups A, B and C each made up of 21 mice and group D comprised 25 mice. Groups A, B and C were infected with 5, 15 and 25 S. japonicum cercariae respectively. As control, mice in group D were left uninfected. 3 mice from each of groups A, B and C were sacrificed and sera collected on days 3, 7, 10, 14, 20, 30, and 60 post infection. All the 25 mice in group D were sacrificed on the first day of the experiment for serum collection. rSjLAP and rSjFBPA were screened and used in ELISA to test the antibody response of the serum samples. Also, sera of 38 acute patients, 96 chronic patients with schistosomiasis japonica, 90 healthy donors and patients with other parasite infections including Clonorchis sinensis (33 cases), Paragonimus westermani (40) and hookworms (37) were tested using the recombinant protein-based ELISA. In addition, 36 sera each from the acute and chronic patients 12 months after treatment with praziquantel and 64 of the chronic patients in more than 2 years post-treatment of praziquantel were tested. The dosage of praziquantel for both acute and chronic patients was 60 mg/kg, 2 times/dx2 d. RESULTS: IgG antibody response was first detected at day 10 post infection by rSjLAP, rSjFBPA or the combined antigen assay. The mean absorbance (A450) on this day were 0.535 +/- 0.053, 0.595 +/- 0.033, 0.696 +/- 0.104 for group B; 0.548 +/- 0.060, 0.608 +/- 0.063, 0.621 +/- 0.090 for group C; and 0.415 +/- 0.038, 0.455 +/- 0.056, 0.498 +/- 0.077 for group A for rSjLAP, rSjFBPA and the combined assay respectively (P < 0.05). Early antibody level to both antigens was significantly higher in mice infected with 15 or 25 cercariae than those with 5 cercariae (P < 0.05). However, ELISA results in patients with confirmed schistosomiasis revealed positive rates of 97.4% (37/38) and 87.5% (84/96) for acute and chronic schistosomiasis with rSjLAP , 94.7% (36/38) and 88.5% (85/96) for acute and chronic schistosomiasis with rSjFBPA and 94.7% (36/38)and 85.4%(82/96) with both rSjLAP and rSjFBPA respectively. Statistical analysis showed no significant difference in the positive rate (P > 0.05). Also, rSjLAP and combined antigens showed a specificity of 96.7% (87/90) while that of rSjFBPA was 97.8% (88/90). There was a general decrease in the antibody titer of the patients after treatment. In 12 months after treatment it was 0.236 +/- 0.212 with rSjLAP, 0.287 +/- 0.191 with rSjFBPA, and 0.235 +/- 0.120 with both antigens respectively for acute cases; For chronic patients, it was 0.266 +/- 0.124, 0.261 +/- 0.143 and 0.265 +/- 0.140 in 12 months post-treatment, and 0.204 +/- 0.074, 0.176 +/- 0.074, and 0.176 +/- 0.073 in 2 years, respectively. For healthy control, it was 0.188 +/- 0.056, 0.173 +/- 0.45, and 0.184 +/- 0.051, respectively. No significant difference on antibody titer was found between treated patients and control (P > 0.05). The cross reaction with C. sinensis was 15.2% (5/33) for rSjLAP, 12.1% (4/33) for rSjFBPA and 9.2% (3/33) for combined antigens. With P. westermani, it was 15.0% (6/40), 12.5% (5/40) and 15.0% (6/40), respectively, and 8.1% (3/37) with hookworm infection. CONCLUSION: The study showed a satisfactory sensitivity and specificity of rSjLAP and rSjFBPA by ELISA which is promising for the immunological diagnosis of schistosomiasis.

Differential expression of TgMIC1 in isolates of Chinese 1 Toxoplasma with different virulence.

BACKGROUND: The predominant genotype of Toxoplasma in China is the Chinese 1 (ToxoDB#9) lineage. TgCtwh3 and TgCtwh6 are two representative strains of Chinese 1, exhibiting high and low virulence to mice, respectively. Little is known regarding the virulence mechanism of this non-classical genotype. Our previous RNA sequencing data revealed differential mRNA levels of TgMIC1 in TgCtwh3 and TgCtwh6. We aim to further confirm the differential expression of TgMIC1 and its significance in this atypical genotype. METHODS: Quantitative real-time PCR was used to verify the RNA sequencing data; then, polyclonal antibodies against TgMIC1 were prepared and identified. Moreover, the invasion and proliferation of the parasite in HFF cells were observed after treatment with TgMIC1 polyclonal antibody or not. RESULTS: The data showed that the protein level of TgMIC1 was significantly higher in high-virulence strain TgCtwh3 than in low-virulence strain TgCtwh6 and that the invasion and proliferation of TgCtwh3 were inhibited by TgMIC1 polyclonal antibody. CONCLUSION: Differential expression of TgMIC1 in TgCtwh3 and TgCtwh6 may explain, at least partly, the virulence mechanism of this atypical genotype.

Toxoplasma gondii Chinese I genotype Wh6 strain infection induces tau phosphorylation via activating GSK3ß and causes hippocampal neuron apoptosis.

Toxoplasma gondii (T. gondii) is a neurophilic and intracellular parasite that can affect plenty of vertebrate animals, including humans. Recent researches indicate that T. gondii infection is associated with neurodegenerative diseases such as Alzheimer's disease(AD). In addition, tau hyper-phosphorylation is a crucial event leading to the formation of nerve fiber tangles in AD. Despite the efforts to understand the interactions between T. gondii and AD, there are no clear results available so far. Here, we infected mice with the T. gondii of the Chinese 1 genotype Wh6 strain (TgCtwh6) for 60 days. Then we observed the formation of tissue cysts in the brain, the damage of neuron and the increased expression of phosphorylated tau (p-tau) in the hippocampal tissue of the mice. Similarly, we also found that p-tau, glycogen synthase kinase 3 beta (GSK3ß), and phosphorylated GSK3ß (p-GSK3ß) were upregulated in vitro in TgCtwh6 challenged hippocampal neuron cell strain, HT22 cells. We noted a down-regulated expression of GSK3ß,p-GSK3ß, and p-tau in HT22 cells, which were pretreated with LiCl, an inhibitor of GSK3ß. These data suggested that p-GSK3ß may mediate tau phosphorylation after TgCtwh6 infection. Furthermore, TgCtwh6 infection also caused the increased expression of Bax and Caspase3, the decreased expression of Bcl-XL in HT22 cells, which had both early and late apoptosis. In all, our results indicated that TgCtwh6 infection not only led to phosphorylation of tau via activating GSK3ß but also promoted hippocampal neuron apoptosis. Our research may partially reveal the mechanism with which TgCtwh6 induce neurofibrillary pathology.

Protective Effect Against Toxoplasmosis in BALB/c Mice Vaccinated With Toxoplasma gondii Macrophage Migration Inhibitory Factor.

Toxoplasma gondii is an obligate intracellular parasite responsible for toxoplasmosis, which can cause severe disease in the fetus and immunocompromised individuals. Developing an effective vaccine is crucial to control this disease. Macrophage migration inhibitory factor (MIF) has gained substantial attention as a pivotal upstream cytokine to mediate innate and adaptive immune responses. Homologs of MIF have been discovered in many parasitic species, and one homolog of MIF has been isolated from the parasite Toxoplasma gondii. In this study, the recombinant Toxoplasma gondii MIF (rTgMIF) as a protein vaccine was expressed and evaluated by intramuscular injection in BALB/c mice. We divided the mice into different dose groups of vaccines, and all immunizations with purified rTgMIF protein were performed at 0, 2, and 4 weeks. The protective efficacy of vaccination was analyzed by antibody assays, cytokine measurements and lymphoproliferative assays, respectively. The results obtained indicated that the rTgMIF vaccine elicited strong humoral and cellular immune responses with high levels of IgG antibody and IFN-γ production compared to those of the controls, in addition to slight higher levels of IL-4 production. After vaccination, a stronger lymphoproliferative response was also noted. Additionally, the survival time of mice immunized with rTgMIF was longer than that of the mice in control groups after challenge infection with virulent T. gondii RH tachyzoites. Moreover, the number of brain tissue cysts in vaccinated mice was reduced by 62.26% compared with the control group. These findings demonstrated that recombinant TgMIF protein is a potential candidate for vaccine development against toxoplasmosis.

[Effect of paeoniflorin on hepatic immunopathogenesis in mice with Schistosoma japonicum infection].

OBJECTIVE: To investigate the mechanism of paeoniflorin in preventing hepatic granuloma formation and fibrosis in mice infected with Schistosoma japonicum. METHODS: Model of hepatic granuloma and fibrosis was established by infecting mice with S. japonicum cercariae. The infected mice were randomly divided into 4 groups: group A as model (infected control) group (15 mice), and paeoniflorin being given before, simultaneously and after praziquantel treatment as groups B, C and D. Each of the groups B, C and D was subdivided into 3 subgroups (15 mice each): low dose (paeoniflorin 2 ml, 30 mg/(kg x d) x 30 d), high dose(paeoniflorin 2 ml, 120 mg/(kg x d) x 30 d) and control (2 ml, 0.5% sodium carboxymethylcellulose x 30 d). In group B, paeoniflorin or sodium carboxymethylcellulose was orally administrated on 12 d after infection. In groups C and D, paeoniflorin or sodium carboxymethylcellulose was administrated on 42 d or 72 d after infection. Each of group B, C and D was orally given praziquantel 2 ml (500 mg/(kg x d) x 2 d) on 42 d after infection. On the 102nd day after infection, all animals were sacrificed by cervical dislocation. Serum hyaluronic acid (HA) was detected by radioimmunoassay; area of egg granuloma and degree of hepatic fibrosis were observed via HE and Masson stainings; the expression of transforming growth factor beta1 (TGF-beta1), alpha smooth muscle actin (alpha-SMA and collagen I (Col I) protein were measured by immunohistochemical method. RESULTS: In group B, the level of HA (0.719 +/- 0.239 microg/ml, 0.721 +/- 0.182 microg/ml) in low or high dose subgroups was significantly lower (F = 9.429, P < 0.01) than the control subgroup (1.049 +/- 0.286 microg/ml); the area of granuloma (0.066 +/- 0.005 mm2, 0.064 +/- 0.004 mm2) or the degree of hepatic fibrosis (2.067 +/- 0.458, 1.967 +/- 0.399) in low or high dose subgroups was significantly greater (F = 862.540, F = 29.738, P < 0.01) than the control (0.141 +/- 0.008 mm2, 3.467 +/- 0.834); the expression of alpha-SMA positive cells (2.933 +/- 0.594, 3.000 +/- 0.535) in low or high dose subgroups was significantly lower (F = 12.323, P < 0.01, P < 0.01) than its control (4.800 +/- 1.859); the expression of TGF-beta1 (0.256 +/- 0.057, 0.274 +/- 0.054) in low or high dose subgroups was significantly lower (F = 148.990, P < 0.01) than its control (0.552 +/- 0.047); the content of Col I (0.334 +/- 0.041, 0.339 +/- 0.042) in low or high dose subgroups was significantly lower (F = 180.881, P < 0.01) than its control (0.601 +/- 0.049). In groups C & D, no significant difference was found between the low or high dose subgroups or between the subgroups and their corresponding controls. CONCLUSION: Paeoniflorin can significantly reduce hepatic granuloma formation and fibrosis due to schistosome eggs, and decrease the expression of TGF-beta1, alpha-SMA in mice when it is given before praziquantel administration, which may associate with the activation of hepatic stellate cells and the expression of TGF-beta1 in liver tissue.

[Effect of paeoniflorin on secretion of TGF-beta1 from macrophages in mice].

OBJECTIVE: To explore the effect of paeoniflorin (PAE) on the production of transforming growth factor beta1 (TGF-beta1) from peritoneal macrophages(PMs) stimulated by soluble egg antigen (SEA) of Schistosoma japonicum. METHODS: SEA was prepared by trituration and added into culture plank, flask and dish containing PMs which were cultured for 24 h. TGF-beta1 secreted from PMs was measured by ELISA. TGF-beta1 mRNA and protein produced from PMs were evaluated by RT-PCR and Western blotting, respectively. SEA (10 mg/L) 5 ml was added into culture flask and dish containing PMs. PMs were cultured for 12 h, and PAE at different concentrations (0, 7.5, 15, 30, 60, 120 mg/L) was added into the culture flask and dish, and PMs were cultured consecutively for another 12 h and 24 h, respectively. TGF-beta1 mRNA and protein from PMs stimulated by SEA were evaluated by RT-PCR and Western blotting, respectively. RESULTS: TGF-beta1 (235.86 +/- 3.43 ng/L) was produced from PMs under stimulation of SEA at 10 mg/L, and the expression of TGF-beta1 mRNA and protein in PMs were depressed significantly by PAE in a concentration-dependent manner (r = -0.827, P < 0.01; r = -0.952, P < 0.01, respectively). CONCLUSION: PAE inhibits the production of TGF-beta1 from PMs stimulated by SEA.

Effective Amelioration of Liver Fibrosis Through Lentiviral Vector Carrying Toxoplasma gondii gra15II in Murine Model.

Our previous investigations indicated that in vitro polarization of mouse macrophages by Toxoplasma gondii type II strain dense granule protein 15 (GRA15 II ), one of the genotype-associated effectors of T. gondii, induced the phenotypes of classically activated macrophage (M1). Transfusion of the cells to mice may effectively alleviated hepatic fibrosis caused by schistosomiasis. The purpose of the study was to identify whether liver macrophages can be in vivo driven to M1 macrophages by lentiviral vector (LV) carrying GRA15 II gene (LV-gra15 II ) and to explore the potential mechanism by which the LV-gra15 II -activated liver macrophage (LV-gra15 II -M) ameliorates the hepatic fibrosis in schistosomiasis. The mice were treated with LV-gra15 II by hydrodynamic injection via the tail vein followed by challenge of Schistosoma japonicum (S. japonicum). Our experiments showed that LV-gra15 II was successfully delivered to liver macrophages and GRA15II was persistently expressed in the macrophages of mice for at least 2 months. Furthermore, the LV-gra15 II infected macrophages were polarized to M1 macrophages in vivo. Consequently, mice with schistosomiasis receiving LV-gra15 II injection displayed a remarkable amelioration of liver granuloma formation and collagen deposition in association with downregulated expression of transforming growth factor-beta1, arginase 1 (Arg-1), α-smooth muscle actin, and an increased expression of matrix metalloproteinase 13 (MMP13). Simultaneously, no negative effects of liver function and vitality of mice were noted. The in vitro experiments indicated that the C-C motif chemokine ligand 2 and nitric oxide level were elevated in LV-gra15 II -M cultural supernatants; hepatocyte growth factor expression was enhanced in LV-gra15 II -M. In addition, LV-gra15 II -M not only secreted MMP13, which greatly degraded type I collagen, but also induced murine hepatic stellate cell (HSC) line (JS1) apoptosis in the co-culture system. Taken together, we identified for the first time that LV-gra15 II may in vivo drive liver macrophages to M1 macrophage phenotypes, which helps for alteration of the liver fibrotic microenvironment with collagen dissolution, HSC deactivation, apoptosis and hepatocyte protection. Our study gives an insight into the use of gene delivery with parasite-derived immunomodulatory factor as a potential immune cell activating agent to re-equilibrate the other pathogen-induced immune response in some chronic diseases.

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.

[Secreted expression of signaling protein 14-3-3 of Schistosoma japonicum in Pichia pastoris system with primary evaluation on its antigenicity].

OBJECTIVE: To express signaling protein Sj14-3-3 in Pichia pastoris and compare its antigenicity with prokaryotic expression one. METHODS: Sj14-3-3 gene was amplified from pET28a-Sj14-3-3 recombinant plasmid, cloned into vector pMD18-T followed by sequencing. The Sj14-3-3 gene was subcloned into the expression vector pPICZalpha-B and transformed into Pichia pastoris X-33 by electroporation. The transformants were identified by sequencing. Three transformants with high copies were obtained when selected under zeocin, and expression was induced with methanol. The culture supernatant was collected and tested by SDS-PAGE and Western blotting. The specificity and sensitivity of eukaryotic expression rSj14-3-3 in Pichia pastoris were compared with that from prokaryotic expression by detecting sera of patients with schistosomiasis by indirect ELISA. RESULTS: The Sj14-3-3 gene was integrated into Pichia pastoris, and the gene of interest detected by PCR was with 1 300 bp. After induction by methanol, the Sj14-3-3 gene was expressed and secreted into the medium. The molecular weight of the recombinant protein was determined as about Mr 35 000 by SDS-PAGE. Western blotting showed that the protein has a high specificity against mouse-anti-Sjl4-3-3 monoclonal antibody. The recombinant protein had a promising immune reactivity. Indirect ELISA showed that by using eukaryotic expression rSj14-3-3 in Pichia pastoris, the positive rate in 36 cases of acute schistosomiasis was 81%, with no cross-reactivity in 12 cases of Clonorchis sinensis, 9.3% cross-reactivity in 32 cases of normal sera. While using prokaryotic expression rSj14-3-3 in E.coli, the positive rate in 36 cases of acute schistosomiasis was 88.9%, with 16.7% cross-reactivity in 12 cases of Clonorchis sinensis, 12.5% cross-reactivity in 32 cases of normal sera. There was no statistically significant difference of the results (P>0.05). CONCLUSION: The recombinant protein Sj14-3-3 of eukaryotic expression in Pichia pastoris has been successfully harvested and shows a promising immunological potential.

Human thelaziosis--a neglected parasitic disease of the eye.

The oriental eyeworm, Thelazia callipaeda (Spirurida, Thelaziidae), infects a range of definitive hosts, such as dogs, cats, foxes, rabbits, and humans. This parasite usually lives under the nictitating membrane of the eye, where the adult females release first-stage larvae into the lachrymal secretions; these larvae are subsequently ingested by the intermediate arthropod host within which they develop to the infective, third-stage larvae. The latter larvae are then deposited into the eyes of the definitive host. Recently, T. callipaeda has been reported to infect dogs, foxes, and/or cats in Europe (Italy, France, and Germany). Human thelaziosis (HT) is considered to be an underestimated parasitic disease, whose prevalence appears to have increased in poor socioeconomic settings in many Asian countries, including China. In humans, the disease can be subclinical or symptomatic, exhibiting epiphora, conjunctivitis, keratitis, excessive lachrymation, corneal opacity, and/or ulcers. Knowledge about HT is presently fragmentary and mainly limited to clinical case reports. This article provides a background on the parasite and its life cycle, reviews cases of human thelaziosis, summarizes key aspects regarding the diagnosis of thelaziosis, and proposes future research and methods of control of the disease in humans, particularly in Asia.

[Progress in researches on macrophages in liver fibrosis of schistosomiasis].

Schistosomiasis causes the imbalance of fibrogenesis and pro-fibrinolytic promoting factors, leading to extracellular matrix deposition and liver fibrosis. The activation of liver macrophages (Kupffer cells) and stellate cells are the two crucial events, which constitute a complex network regulating fibrosis balance. This review discusses the function of fibrotic cytokines secreted by macrophages, and their interaction and mutual influence with stellate cells in hepatic fibrosis. Additionally, the expected progress and novel in vitro and in vivo approaches that have been achieved recently in our laboratory are briefly introduced.