In Vitro Virulence Contrast of Seven Genetically Distinct Toxoplasma gondii Isolates After Rejuvenation In Vivo.

BACKGROUND: In the past for more than 100 years at least 300 genotypes of Toxoplasma gondii were recorded and several traditional isolates such as RH, GT1, ME49, PRU and VEG were used repeatedly to clarify the pathogenic mechanisms and the epidemiological significance to human, but for if their virulence was mutative post-iterative passages it remains confused. OBJECTIVE: Therefore, in the study, seven genetically distinct T. gondii including C7 and PYS previously discovered in China were reidentified by sequencing the head of hsp40 locus to distinguish their virulence in vitro post-rejuvenation in vivo. RESULTS: Our data showed the nucleotides were different in 18 positions and 7 of them can be used to type T. gondii isolates. Total 634 plaques of T. gondii without two or more overlaps indicated that RH and GT1 tachyzoites possess stronger power than other five isolates in vitro (p < 0.001), followed by ME49, PRU, C7, PYS, and the weakest VEG. Based on the shapes of plaques, we found the ratio of their width/length was associated with the virulence of T. gondii, and speculated it could be used to judge T. gondii tachyzoites in vitro, whereas the data of simple linear regression analyses did not agree. CONCLUSIONS: Together, virulence of seven genetically distinct T. gondii isolates that can be distinguished by seven mutative nucleotides in hsp40 was redefined in vitro post-rejuvenation in vivo, and it cannot be directly judged just according to the shapes of plaques formed in vitro.

Ultrasound features affecting the sample adequacy after fine-needle aspiration of thyroid nodules with different risk stratification.

BACKGROUND: The inadequacy samples caused by the internal characteristic structure of thyroid nodules are difficult to be solved. OBJECTIVE: To evaluate the ultrasound features affecting the sample adequacy after fine-needle aspiration (FNA) of thyroid nodules with different risk stratification. METHODS: 592 thyroid nodules that underwent ultrasound-guided FNA were included in this retrospective study. The sample obtained by FNA were classified as inadequacy and adequacy according to the cytopathological results. Ultrasound features (ie., size, position, cystic predominance, composition, echo, shape, margin, and superficial annular calcification status) of the nodules were recorded and compared between the inadequacy sample group and adequacy sample group. RESULTS: Multiple logistic regression shows that preponderant cystic proportion (OR, 0.384; P = 0.041), extremely hypoechogenicity and hypoechogenicity (OR, 6.349; P = 0.006) were the independent influencing factors of inadequate samples after FNA in benign expected nodules. In addition, nodule size ≤10 mm (OR, 1.960; P = 0.010) and superficially annular calcification (OR, 4.600; P < 0.001) were independent influencing factors for inadequate samples after FNA in malignant expected nodules. CONCLUSION: The ultrasound features of hypoechogenicity or high cystic proportion in benign expected nodules and that of small size or annular calcification in malignant expected nodules were the risk factors for inadequacy samples by US-guided FNA.

Immunization of BALB/c Mice with Killed Tachyzoites of Toxoplasma gondii against Acute Toxoplasmosis.

Background: Toxoplasma gondii with widespread distribution infects over one third of human populations in the world and can cause serious life-threatening diseases especially for the immunodeficient patients in acute toxoplasmosis. As the clinical pharmaceutical drugs with severe side effects for treatment and non-ideal extant vaccines for prevention, more work starves to be done for keeping advantages in the athletics. Methods: Aluminum adjuvant and hybrid formaldehyde-killed tachyzoites of T. gondii RH and GT1 isolates were prepared to intramuscularly immunize BALB/c mice for five times at 0, 3, 7, 14 and 21 days post first injection. The triggered humoral and cellular immune responses at two weeks post the last immunization and the survival times of infected mice were examined for the hybrid immunization scheme judgement. Results: The anti-RH and anti-GT1 specific antibodies were both increased at one week prior to challenge (P < 0.05), and the survival times of immunized mice (7.33 ± 0.71 d for RH, 7.22 ± 0.97 d for GT1) against acute toxoplasmosis were significantly prolonged by the immunizations performed in the study compared to blank control (6.67 ± 0.50 d for RH, 6.33 ± 0.71 d for GT1; P < 0.05), with the higher IFN-γ, IL-2 and IL-12p70 in sera, the elevated CD3e+CD4+ T and CD3e+CD8a+ T cells, and the enhanced lymphocyte proliferation in spleen (P < 0.05). Conclusion: The hybrid killed tachyzoites with aluminum adjuvant induced humoral and cellular immune responses of mice, and offered mildly protective efficacy against acute toxoplasmosis.

Distinctive distribution and migration of global fallout plutonium isotopes in an alpine lake and its implications for sediment dating.

This work investigated plutonium (Pu) isotopes in sediment cores collected from an alpine lake (Lake Heinongpo with 3779 m above sea level) in Southwestern China. 240Pu/239Pu atom ratios in all sediment samples showed the typical global fallout values of ∼0.18 without any influences from other Pu contaminant sources. 239+240Pu activities with surface and subsurface maximums followed by exponential decline with sediment depth were respectively observed in the two sediment cores. The distinctive depth distributions of 239,240Pu in the lake sediments was attributed to the very slow sediment deposition rate due to the lack of terrestrial sediment input, while the alpine snowmelt input was the primary source of Pu in the lake sediments in addition to the direct atmospheric deposition. The total Pu inventory was estimated to be 56.3 ± 1.4 and 63.9 ± 0.8 Bq/m2 respectively in the two sediment cores. The generally higher Pu inventory in the Lake Heinongpo compared with other reported lakes in similar latitude should be mainly attributed to their different Pu input passages. The advection-diffusion equation was further applied to evaluate the downward migration of Pu isotopes in the sediment cores and predict the future evolution of Pu distribution in the sediment cores. The fitted results indicated that the diffusion effect controlled the downward migration of Pu in the sediments, but this diffusive migration will not prevent the peak of global fallout Pu in undisturbed sediment cores from being a valuable time marker for sediment dating.

Toxoplasma invasion delayed by TgERK7 eradication.

Toxoplasma gondii causes serious clinical toxoplasmosis in humans mostly due to its asexual life cycles, which can be artificially divided into five tightly coterminous stages. Any radical or delay for the stage will result in tremendous changes immediately behind. We previously demonstrated that TgERK7 is associated with the intracellular proliferation of T. gondii, but during the process, other stages before were not meanwhile determined. To further clarify the function of ERK7 gene in T. gondii, the complemental strain of ΔTgERK7 tachyzoites created previously was engineered via electric transfection with the recombinant pUC/Tgerk7 plasmid, named pUC/TgERK7 strain in this study, and was used together with ΔTgERK7 and wild-type GT1 strains to retrospect the phenotypic changes including invasion and attachment. The results showed that TgERK7 protein can be re-expressed in the ΔTgERK7 tachyzoites and eradication of this protein leads to significantly lower invasion of T. gondii at 1 h and 2 h post-infection (P < 0.05), which is the key factor causing the following slow intracellular proliferation, in comparison with wild-type GT1 and pUC/TgERK7 parasites; noteworthily, at other early time points including 15 min for attachment assay was no statistical difference (P > 0.05). The data suggested that ERK7 protein in T. gondii is an important virulence factor that participates in the invasion of this parasite.

Immunostimulatory efficacy and protective potential of putative TgERK7 protein in mice experimentally infected by Toxoplasma gondii.

The extracellular signal-regulated kinases (ERKs) serve as important determinants of cellular signal transduction pathways, and hence may play important roles during infections. Previous work suggested that putative ERK7 of Toxoplasma gondii is required for efficient intracellular replication of the parasite. However, the antigenic and immunostimulatory properties of TgERK7 protein remain unknown. The objective of this study was to produce a recombinant TgERK7 protein in vitro and to evaluate its effect on the induction of humoral and T cell-mediated immune responses against T. gondii infection in BALB/c mice. Immunization using TgERK7 mixed with Freund's adjuvants significantly increased the ratio of CD3e+CD4+ T/CD3e+CD8a+ T lymphocytes in spleen and elevated serum cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-12p70, IL-23, MCP-1, and TNF-α) in immunized mice compared to control mice. On the contrary, immunization did not induce high levels of serum IgG antibodies. Five predicted peptides of TgERK7 were synthesized and conjugated with KLH and used to analyze the antibody specificity in the sera of immunized mice. We detected a progressive increase in the antibody level only against TgERK7 peptide A (DEVDKHVLRKYD). Antibody raised against this peptide significantly decreased intracellular proliferation of T. gondii in vitro, suggesting that peptide A can potentially induce a protective antibody response. We also showed that immunization improved the survival rate of mice challenged with a virulent strain and significantly reduced the parasite cyst burden within the brains of chronically infected mice. Our data show that TgERK7-based immunization induced TgERK7 peptide A-specific immune responses that can impart protective immunity against T. gondii infection. The therapeutic potential of targeting ERK7 signaling pathway for future toxoplasmosis treatment is warranted.

Devitalization of the immune mapped protein 1 undermines the intracellular proliferation of Toxoplasma gondii.

The intracellular protozoan Toxoplasma gondii infects approximately one-third of the world's population as well as various animals, causing toxoplasmosis. However, there remains a need to define the functions of newly identified genes of T. gondii. In the present study, a novel molecule, immune mapped protein 1 of T. gondii (TgIMP1), was devitalized by CRISPR/Cas9 system to investigate the phenotypic changes of the parasite. We found that the virulence of ΔTgIMP1 knockout strain was reduced in comparison with wild-type GT1 tachyzoites, showing a statistically decreased plaque in HFF cells and a significantly prolonged survival period of mice (P < 0.05). Moreover, the data of phenotype analyses in vitro showed a different level of the intracellular proliferation and the subsequent egress between ΔTgIMP1 and wild-type GT1 strain (P < 0.05); while no statistically significant difference was detected during the process of attachment or invasion. These results suggested that TgIMP1 is closely associated with the intracellular proliferation of this parasite.

Immune Responses Induced by pVAX/TgERK7 against Toxoplasma gondii Infection in BALB/c Mice.

BACKGROUND: Toxoplasma gondii can infect all the warm-blooded vertebrates and cause serious toxoplasmosis. Extracellular signal-regulated kinase 7 in T. gondii (TgERK7) mediated the proliferation of this parasite may be a potential vaccine candidate. Thus, immune responses induced by TgERK7 were investigated in this study using a DNA vaccine strategy. METHODS: pVAX/TgERK7 plasmid was constructed and used to immunize BALB/c mice for three times with two-week intervals. The challenge and the investigation of humoral and cellular immune responses were performed at two weeks post the last immunization, and the survival times of the infected mice were daily recorded until all of them were dead. RESULTS: The innate immune response with higher concentrations of IFN-γ, TNF-α, IL2 and IL12p70 in sera (P < 0.05), and the adaptive immune responses were evoked by the DNA immunizations, including specific antibody, lymphocyte proliferation, and the CD3e+CD4+ and CD3e+CD8a+ T cell-mediated response effects. Interestingly, no significant difference was detected in their survival times among all the experimental groups of mice that were challenged with GT1 tachyzoites or PRU cysts (P>0.05). CONCLUSION: The successive immunizations with pVAX/TgERK7 can provoke the innate and adaptive immune responses of BALB/c mice, whereas the DNA vaccine-induced immunological efficacy is not sufficient for complete protection the host against T. gondii infection.

Immunization with plasmid DNA expressing Heat Shock Protein 40 confers prophylactic protection against chronic Toxoplasma gondii infection in Kunming mice.

Toxoplasma gondii causes one of the most common protozoal diseases of humans and animals worldwide. With the aim of designing an effective vaccine against T. gondii infection, we examined the immunogenicity of a DNA vaccine expressing heat shock protein 40 (HSP40) against challenge with T. gondii (type I RH and type II Pru) strains in Kunming mice. The plasmid pVAX1-HSP40 was constructed and used to immunize mice by intramuscular injection for three sequential immunizations with two-week intervals. This immunization regimen significantly reduced parasite cyst burden in pVAX1-HSP40-immunized mice (1871.9 ± 142.3) compared with control mouse groups immunized with pVAX1 (3479.2 ± 204.4), phosphate buffered saline (3024.4 ± 212.8), or left untreated (3275.0 ± 179.8) as healthy controls (p < 0.01). However, immunization failed to protect mice against challenge with the virulent RH strain. There was a significant increase in T lymphocyte subclasses (CD3e+CD4+ T and CD3e+CD8a+ T lymphocytes) in splenic tissues in immunized mice compared with controls (p < 0.05). However, the level of antibodies, lymphocyte proliferation and concentration of cytokines (IFN-γ, IL-2, IL-4, IL-10 and IL-12p70) were not significantly different between immunized and control mouse groups (p < 0.05). These data indicate that pVAX1-HSP40 induced specific immune responses and achieved a significant reduction in the number of brain cysts in Pru-infected mice, and thus can be tested in future immunization studies along with plasmids containing other immunogenic proteins as a cocktail vaccine to fully abolish chronic toxoplasmosis.

Sources of plutonium isotopes and 137Cs in coastal seawaters of Liaodong Bay and Bohai Strait, China and its environmental implications.

In order to investigate the sources of plutonium in seawaters of Liaodong Bay and Bohai Strait, China, surface seawater samples were collected and analyzed for 239+240Pu and 137Cs by radiochemical separation combined with ICP-MS and γ-spectrometry, respectively. A large variation of 239+240Pu activities was observed, ranging from 1.993 to 29.677 mBq/m3 in the Liaodong Bay and from 0.932 to 10.183 mBq/m3 in the Bohai Strait. 137Cs activities showed little variation between the investigated locations, which was attributed to the different behavior characteristics of 239+240Pu and 137Cs in seawaters. The 240Pu/239Pu atom ratios showed a significant variation between the Liaodong Bay (0.185) and Bohai Strait (0.225), indicating that Pu derived from the Pacific Proving Grounds might have been transported to the Bohai Strait but not yet to the Liaodong Bay, which could further provide valuable reference for evaluating the seawater exchange cycle between the Bohai Sea and Yellow Sea.

[Analysis of the storage time in paraformaldehyde-fixed T cells and their subsets in Kunming mice].

Objective To determine the expiration dates of CD3e+CD4+ T and CD3e+CD8a+ T lymphocytes of Kunming mice. Methods The sterile spleen and anticoagulant blood of Kunming mice were collected and used to prepare the single-cell suspensions. After CD3e, CD4 and CD8a of T cells were stained with fluorophore-conjugated monoclonal antibodies, the samples were fixed with paraformaldehyde (PFA) and stored at 4DegreesCelsius in dark followed by detection using three-color flow cytometry at several specific time points. The best regression curves fitted with the least squares method were used for calculating the contents of T cells at each time point and the subsequent statistical analysis. Samples without PFA served as the blank controls. Results The levels of PFA-fixed CD3e+CD4+ T and CD3e+CD8a+ T lymphocytes from spleen tissues significantly decreased at 11 days and 16 days post collection, respectively; whereas no difference was found in the samples without PFA until 8 days and 11 days post collection, respectively. As for the T cells separated from the peripheral blood, the longer storage periods were both 18 days under the PFA fixation, which were longer than those of the blank controls (10 and 15 days, respectively). Conclusion The PFA-fixed T lymphocytes of the spleen and peripheral blood of Kunming mice should be detected using flow cytometry within 11 days post collection.

Intervention of childhood and adolescents obesity in Shantou city.

OBJECTIVES: The purpose of this study was to evaluate the effects of multicomponent school based intervention constituted of diet modification, regular exercise and psychosocial consultation on body status in overweight and obese children and adolescents. And to come up with an appropriate intervention protocol for controlling children and adolescents obesity in Shantou city. METHODS: Two schools were randomised to intervention group and control group respectively. A total of 41 students enrolled were diagnosed as overweight or obese. Twenty-six students of the intervention group completed the one-year intervention programme consisted of diet modification, regular exercise and psychological consultation except two of them were transferred to another school. The differences of BMI, anthropometric measures, metabolic profile and the scores of questionnaire and the scale were compared to evaluate the effects of the intervention programme. RESULTS: After one-year intervention, it observed in the intervention group that BMI-Z score, WHR and WHtR had significant improvements, and there was a nonsignificant trend (P=0.053) for a decrease in BMI-P. Fasting plasma glucose, cholesterol (CH) and low-density-lipoprotein cholesterol (LDL-C) levels in the intervention group showed nonsignificant trend for a decrease (PFPG=0.084, PCH=0.057, PLDLC=0.098), compared with a significant increase of triglycerides (TG) and LDL-C levels in the control group (PTG=0.041, PLDL-C=0.038). There were some positive dietary, physical activity, or sedentary behaviour changes found in the students of the intervention group as the scores of the questionnaire got significant improvement (P=0.04). CONCLUSIONS: Our one-year multicomponent school-based intervention programme did have positive effects to some extents on health state and lifestyle behaviour of overweight and obese children and adolescents, which indicated that it is feasible and important to implement such a school-based intervention programme in Shantou city.