The 150-kDa oxygen-regulated protein (ORP150) regulates proteinuria in diabetic nephropathy via mediating VEGF.

The 150-kDa oxygen-regulated protein (ORP150) belongs to a family of the heat shock protein implicated in the cellular response to environmental stress. Previous data demonstrated that ORP150 regulates the secretion of vascular endothelial growth factor (VEGF) to drive progression of angiogenesis associated with proliferative diabetic retinopathy. However, the expression and biological functions of serum ORP150 levels in diabetic nephropathy (DN) remain unclear. In this study, we reported for the first time that ORP150 was up-regulated in serum of patients with DN. Moreover, we observed the dramatic increase in serum ORP150 accompanied with the elevated levels of proteinuria and serum VEGF levels in DN, indicating the possible involvement of ORP150 in regulation of albuminuria via mediating VEGF in DN. Employing the streptozotocin (STZ) to construct the DN model, we confirmed the positive correlation of ORP150 with VEGF in vivo. Monoclonal anti-ORP150 antibodies treatment significantly decreased the secretion of VEGF and albuminuria in STZ-induced DN models. Consequently, our data suggested that ORP150 levels were positively correlated with proteinuria burden via mediating VEGF in DN. It may be considered as a novel diagnostic and therapeutic target.

[The role of Th17/Treg imbalance in liver fibrosis in schistosomiasis].

Liver fibrosis in schistosomiasis is a serious pathological consequence from immune reactions to schistosome infection. The progression of liver fibrosis depends on the state of immune response. Recent studies have found that Th17 and Treg cells are two subsets of CD4+T cells. The Th17 cells are mainly involved in inflammatory responses, while the Treg cells mainly mediate downregulation of the responses. Under normal conditions, the differentiations of the two subsets are inhibited by each other, and they function oppositely. The balance between Th17 and Treg cells, as well as the balance between them, play an important role in the maintenance of homeostasis and are involved in inflammatory responses, tissue trauma, fibrosis and development of many diseases. This paper reviews the role of Th17/Treg cells and their imbalance in liver fibrosis in schistosomiasis.

[Effect of Toxoplasma gondii Infection in Female Mice on Dopamine Level in the Brain of the Male Offspring].

OBJECTIVE: To investigate the effect of Toxoplasma gondii infection in female mice on dopamine level in the brain of male offspring. METHODS: Thirty-six ICR female mice were randomly divided into control group and infection group, 18 mice in each group. Each mouse in infection group was orally infected with 10 cysts of T. gondii Prugniaud strain. On the 90th day after infection, the infected female mice were mated with normal male ICR mice at 1:1 ratio. On the 20th day of pregnancy, 2 mice in each group were delivered for fetal mice by cesarean section, and the brain of male fetal mice (n = 6) in each group were collected. On the 14th and 63rd day after birth, 6 male offspring mice in each group were sacrificed, and the brain were collected. Dopamine levels in the cortex, cerebellum, hippocampus, and striatum were analyzed by high-performance liquid chromatography-electrochemical detection (HPLC-ECD). RESULTS: Three mice in infection group died during the experiment, and 6 out of 15 female mice mated successfully. The number of fetal mice and F1 generation mice in infection group was 12 (male: 7) and 21 (male: 15), respectively. All the mice in control group mated successfully. The number of fetal mice and F1 generation mice was 23 (male: 12) and 179 (male: 92), respectively. The dopamine level in the cerebellum of fetal mice of infection group and control group was (413.25 ± 21.78) ng/g and (346.30 ± 51.83) ng/g, respectively (P < 0.01). No significant difference was found in dopamine content in the cortex between the two groups (P > 0.05). Compared with the control group, on the 14th day and 63rd day after birth, the dopamine content in cortical areas [(462.50 ± 24.80) ng/g and (1215.77 ± 113.64) ng/g], cerebellum area [(271.55 ± 26.19) ng/g and (1328.82 ± 39.62) ng/g], hippocampus area [(225.78 ± 24.17) ng/g and (1322.70 ± 58.34) ng/g], and striatum area [(455.23 ± 61.53) ng/g and (991.32 ± 54.31) ng/g] of the male offspring in infection group were significantly higher than that of the control (P < 0.05, P < 0.01). CONCLUSION: T. gondii infection in female mice causes an increase of dopamine level in the brain of F1 generation male mice.

[Dynamic changes of sciatic nerve conduction velocity in rats infected with Toxoplasma gondii].

OBJECTIVE: To observe the dynamic changes of sciatic nerve conduction velocity of Toxoplasma gondii-infected rats at different time points. METHODS: Twenty SD rats were randomly divided into control group and Toxoplasma gondii infection group. Rats in T. gondii infection group were intraperitoneal injected with 4x10(7) T. gondii tachyzoites, while those in control group were given equivalent normal saline. Motor and sensory nerve conduction velocities (MNCV, SNCV) in sciatic nerve were measured by Medtronic Keypoint4 Workstation electromyography at pre-infection, and 2, 4, 8, 12 months post-infection. RESULTS: Within two months after infection, there was no difference in SNCV and MNCV between control group and infection group (P>0.05). From 4 months after T. gondii injection, infected rats began to show the slowness of SNCV and MNCV, which progressed with the course of infection. At 4, 8, and 12 months after infection, SNCV and MNCV of infection group were (35.26±3.02) and (25.94±3.20) m/s, (33.57±2.27) and (22.75±2.31) m/s, and (32.38±2.38) and (22.03±2.08) m/s, respectively. Compared with control group, SNCV and MNCV of infection group reduced by (7.47±2.11)% and (12.57±1.89)%, (8.92±2.64)% and (13.72±2.65)%, and (12.18±1.94)% and (15.46±2.37)%, respectively (P<0.05). CONCLUSION: From 4 months after infection, Toxoplasma gondii-infected rats show a slowness of motor and sensory nerve conduction velocities in sciatic nerve.

[Proteomic study of the hippocampus tissue from rats with chronic Toxoplasma gondii infection].

OBJECTIVE: To observe the proteome changes in the hippocampus tissue of rats with chronic Toxoplasma gondii infection. METHODS: Six male SD rats were randomly divided into control group and infection group. Each rat in infection group was intraperitoneally injected with 4 x 10(7) purified T. gondii tachyzoites. Rats in the control group received equivalent volumes of sterile normal saline. At the fifth day post-infection, blood samples were taken from the lateral tail vein and Ciemsa staining of blood cells was performed to find Toxoplasma gondii. Rats were dissected at the 10th week post-infection, total protein in the hippocampus was separated by using two-dimensional gel electrophoresis (2-DE). After Coomassie blue staining, the Image Analysis software was used to select and separate proteins on the gel. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for peptide mass fingerprint PMF). Proteins were identified by using Mascot software to search the MSDB and SwissProt databases. RESULTS: Microscopy examination of blood smears confirmed that the rats in infection group were all infected by 11 gondii. The number of protein spots of rats from infection group and control group was 311 +/- 19 and 327 +/- 13 respectively. Compared with the control group, 5 protein spots disappeared, 4 protein spots were up-regulated and 7 were down-regulated in the infection group. The 9 differentially expressed protein spots were identified by MALDL-TOF-MS: phosphoglycerate kinase 1, similar to alpha-enolase, glutamine synthetase, creatine kinase, creatine kinase B-type, ATP synthase, aconitase 2, mitochondrial precursor, actin and an unnamed protein. The first three proteins were up-regulated and the other five proteins were down-regulated in infection group. CONCLUSION: Nine differential expression proteins are found from the hippocampus tissue in rats chronically infected with T. gondii and normal SD rats.

[Impairment of learning and memory ability in mice with latent infection of Toxoplasma gondii].

OBJECTIVE: To detect the learning and memory ability in mice model of latent Toxoplasma gondii infection with object recognition test and Morris water maze test. METHODS: Thirty-six Kunming mice were divided into control group, infection group with 6 cysts each mouse (low infection group), and infection group with 12 cysts each mouse (high infection group) averaged. Mice in the two infection groups were orally infected with T. gondii Prugniaud (PRU) low virulence strain. Object recognition test was conducted at the 63rd day after infection. After the first day of adaptation and the second day of familiarization in the test, the time expended on exploring new and familiar objects was recorded on the third day and the discrimination index (DI) was calculated. Morris water maze test was conducted at the 66th day. The ability of spatial learning, spatial memory retention and working memory capacity was evaluated by place navigation test, spatial probe test, and working memory test, respectively. The mice were sacrificed at the 74th day after infection. The left cerebral hemisphere of mice was fixed, sliced, and stained with eosin-hematoxylin for pathological examination. The right hemisphere was used to detect the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) content. RESULTS: The results of object recognition test showed that the discrimination index of high infection group and low infection group was (14.3 +/- 5.2)% and (17.5 +/- 5.6)%, respectively, significantly lower than the control [(28.9 +/- 7.1)%] (P < 0.01). In the place navigation test, the latency to find the platform in the two infection groups was longer than the control, with significant difference on the second and third day (P<0.05). In the spatial probe test, the percentage of the distance across the platform quadrant in the total swimming distance of high infection group and low infection group were (19.9 +/- 5.0)% and (23.9 +/- 6.8)%, respectively, significantly lower than the control [(27.4 +/- 3.6)%] (P < 0.05). In the working memory test, at the fourth day of test the latency of high infection group and low infection group [(365 +/- 14.2) s and (35.3 +/- 13.7) s] was significantly longer than the control [(30.4 +/- 12.5) s] (P<0.05). In all the tests, there was no statistical significance between low infection group and high infection group (P > 0.05). The brain sections of two infection groups showed cysts of T. gondii, proliferation of glial cells, widened gap around small blood vessels, and a phenomenon of "vascular cuff". The activity of SOD in the mice brains of two infection groups was significantly lower than the control, while MDA level was significantly higher (P < 0.05). SOD and MDA showed no significant difference between two infection groups (P > 0.05). CONCLUSION: Latent infection of T. gondii may lead to learning and memory impairment in mice.

[In vitro co-cultivation of Toxoplasma gondii tachyzoites with rat brain astrocytes].

Purified astrocytes were cultured in plates. When astrocytes grew over 80% of the plate, tachyzoites of Toxoplasma gondii RH strain were added for co-culture. In the period of 0-72 h, change of the astrocytes and tachyzoites was observed after Giemsa staining. In 0-48 h, monodansylcadaverine (MDC) was used to study the action of autophagy in the process of tachyzoites invading astrocytes. At 1 h co-culture, tachyzoites had entered in astrocytes and the autophagosomes appeared. At 4 h, the autophagosomes increased pronouncedly. However, after 12 h, number of autophagosomes considerably decreased and damage of the cells occurred. 48 h later, autophagosomes disappeared and more astrocytes were destroyed. At 72 h most cells destroyed and tachyzoites were released. The result showed that autophagy is inhibited when the astrocytes were in vitro infected by tachyzoites.

Changes to the gut microbiota in mice induced by infection with Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is a common parasite worldwide, which can cause encephalitis, enteritis and miscarriage in abortion women. This study examined the cecal microbiome of mice infected with T. gondii through analysis of 16S rRNA genes determined by Illumina sequencing. BALB/c mice were orally infected with sporulated T. gondii oocysts. Mice were killed after 13-days- and 21-days- post infection, respectively, then their cecal contents were extracted and examined to determine the composition of gut microflora by illumina sequencing of the V3 +V4 region of the 16S rRNA genes. Our results showed the alterations in the gut microbes of BALB/c mice infected with T. gondii infection, where we observed a significant shift in the relative abundance of cecal bacteria. In mice at 13 days post-infection, the relative abundance of Proteobacteria increased, along with that of harmful bacteria, such as Bilopha and Desulfovibrio. However, the abundance of Lactobacillus decreased. At 21 days post-infection, the abundance of Lactobacillus was more than that observed for the uninfected control, with harmful bacteria, such as Bilopha and Desulfovibrio being reduced. The mice at 21-days post-infection had more beneficial intestinal bacteria than the control group. Our results suggested that the gut microbiota play an important role in disease progression from acute infection to chronic infection.

[Changes of peripheral blood T lymphocytes, IFN-gamma, TNF-alpha and IL-4 in rats infected by Toxoplasma gondii].

OBJECTIVE: To observe the changes of peripheral blood T lymphocytes, IFN-gamma, TNF-alpha and IL.4 in rats infected by T gondii. METHODS: 48 Sprague-Dawley(SD) rats were intra-abdominally injected with 2 x 10(5)/L of cellulose purified living tachyzoites in 2 ml and randomly divided into 8 groups Six rat was intra-abdominally injected 2 ml of saline as control and 4 rats were remained as normal control. Peripheral blood was collected and the level of IFN-gamma, TNF-alpha, IL-4 was analyzed by ELISA on day 1, 3, 7, 14, 28, 35, 42, 60. RESULTS: Level of IFN-gamma (6.73 pg/nil) and IL-4 (6.91 pg/ml) increased in experimental rats on day 7 (P < 0.05) and maintained. Level of TNF-alpha (14.37 pg/ml) increased in experimental rats on day 28 (P < 0.05), and that of CD8+ T lymphocytes (14.22%) was signficantly lower than that in control (23.08%) (P < 0.05) and recovered on day 28. No considerable change was observed on the level of CD4 T lymphocytes. CONCLUSION: The level of CD8 T lymphocytes, IFN-gamma, TNF-alpha and IL-4 in the rat can be affected by the infection of T. gondii and the level of CD4+ T lymphocytes shows no change.

Developing a rat model of dilated cardiomyopathy with improved survival.

To compare the continuous infusion and intermittent bolus injection administration protocols of doxorubicin (Dox) under the same cumulative dose (12 mg/kg), and establish a rat dilated cardiomyopathy model with improved survival, a total of 150 Sprague-Dawley (SD) rats were divided into three groups: a control group, administered with normal saline; a Dox 1 group, administration twice a week at 1 mg/kg; a Dox 2, administration once a week at 2 mg/kg. Mortality rates in the Dox 1 and Dox 2 groups were 22% and 48%, respectively (P<0.05). As shown by echocardiography, both Dox groups exhibited significant chamber dilatation and reduced cardiac function (all P<0.05 vs. control). Plasma brain natriuretic peptide and C-reactive protein concentrations were significantly increased (P<0.05) with both Dox regimens. The concentrations of Caspase-3 in myocardial tissues of rats significantly increased in both doxorubicin regimens. Myocardial metabolism imaging by histology and 18F-fluoro-deoxyglucose-positron emission tomography (18FDG-PET) both revealed decreased myocardial viability and necrosis, and even interstitial fibrosis, in left ventricles (LVs) in both Dox groups. Serum creatinine and aspartate aminotransferase concentrations were significantly higher in the Dox 2 model than in the Dox 1 model. Doxorubicin given at both regimens induced dilated cardiomyopathy, while its administration at lower doses with more frequent infusions reduced the mortality rate.

[Survey of infection of Toxoplasma gondii in infertile couples in Suzhou countryside].

OBJECTIVES: To determine the level of anti-Toxoplasma antibody in serum of infertile couples to explore the relationship between toxoplasma infection and infertility. METHODS: Enzyme-linked immunosorbent assay (ELISA) was applied to detect the anti-Toxoplasma antibody, antisperm antibody (AsAb) and anticardiolipin antibody (ACA) in serum of 178 couples with infertility and 190 couples who had normal pregnant history. RESULTS: The positive result of Toxoplasma infection in the infertile couples was significantly higher than that in fertile couples which was 34.83% vs 12.11% (chi 2 = 26.72, P < 0.01) with the odds ratio 3.88. The positive result of serum AsAb in the Toxoplasma infected group was significantly higher than that in the no Toxoplasma infected group (32.50% vs 15.94%, chi 2 = 10.76, P < 0.01) with the odds ratio 2.54. CONCLUSIONS: Toxoplasma infection was related to infertility. The Toxoplasma infection and was posibly related to the antisperm antibodies which can be involved in the pathogenisis of infertility.

[Observation of animal model of life cycle of Angiostrongylus cantonensis].

OBJECTIVE: To observe the effect of artificially construction of the life cycle of Angiostrongylus cantonensis in the laboratory condition, so as to provide the basis for the research of angiostrongyliasis. METHODS: SD rats were infected orally with the third-stage larvae of A. cantonensis collected from infected Pomacea canaliculata. Six weeks after the infection, the first-stage larvae were isolated and counted from fresh feces of the rats and then were used to infect P. canaliculata. Three weeks later, the snails were dissected for counting the third-staged larvae of A. cantonensis. RESULTS: The first-stage larvae were detected in the feces of the rats 6 weeks after the infection, and the third-staged larvae were successfully isolated after the infection of P. canaliculata. CONCLUSION: The animal model of the entire life cycle of A. cantonensis is successfully established in the laboratory with the infection of 50 larvae per rat.

[Progress of research on microRNAs of parasites].

microRNAs (miRNAs) are small molecules of non-coding RNA, a class size of about 21-25 nt widespread in eukaryotes, resulting from single-stranded RNA precursors with the size of 70-90 bases of a hairpin structure generated after dicer enzyme processing. They play an important role in eukaryotic gene regulation, widely involved in cell proliferation, differentiation, development, metabolism, apoptosis and other physiological activities. miRNA extensively involved in the physiological and metabolic processes of the parasite development process, but the key miRNA relevant to parasite invasion of a host still lacks reports. This paper summarizes the miRNA analytical methods and the progress on its researches in parasitology.

[Seroepidemiological survey of chickens infected with Toxoplasma gondii in Wuxi City].

OBJECTIVE: To understand the status of scattered chickens infected with Toxoplasma gondii in Wuxi City. METHODS: We investigated the positive rate of Toxoplasma antigen and antibody of 309 cases of scattered chickens in Wuxi, and it was compared with the positive rate of Toxoplasma antigen and antibody of 150 cases of intensive farming chickens. RESULTS: In the 309 cases of scattered chickens, there were 29 cases of antigen positive, and the positive rate was 9.39%; there were 53 cases of antibody positive, and the positive rate was 17.15%; there were 13 cases of antigen and antibody double positive, and the double-positive rate was 4.21%.; In the scattered chickens, the overall positive rate was 22.33%, and in the intensive farming chickens, the overall positive rate was 2.67%, and there was a significant difference between them (chi2 = 29.19, P <0.01). CONCLUSION: There is a high T. gondii infection in scattered chickens in Wuxi, so the medical, veterinary and food hygiene workers should pay more attention to it.

[Serological investigation on Toxoplasma gondii infection in patients with unknown central nervous system diseases].

Totally 207 patients with unknown central nervous system diseases and 203 healthy persons were investigated for serum IgG of anti-Toxoplasma antibody assessed by ELISA. The serum IgG positive rate in 207 patients with unknown central nervous system diseases was 19.81%, and that in 203 health people was 5.42%, and there was a significant difference between them (P < 0.01). The IgG positive rates in different types of central nervous system diseases were different, which were 22.81%, 24.32%, 16.05%, and 18.75%, respectively in encephalopathy, epilepsy, mental disorder and neurasthenia. The IgG positive rate in different types of central nervous system diseases were significantly higher than that in healthy population (P < 0.01). The IgG positive rates in patients who contacted or did not contact cats or dogs were 32.97% and 9.48% respectively (P < 0.01). In conclusion, the infection rate in patients with unknown central nervous system diseases is higher than that in healthy persons; therefore, it is necessary to assay the serum IgG in them.

[Preliminary study on serum proteomics in mice with acute toxoplasma gondii infection by using functionalized magnetic beads and MALDI-TOF-MS technique].

OBJECTIVE: To detect and analyze the serum protein biomarkers in mice with acute Toxoplasma gondii infection. METHODS: The serum samples from 8 C57BL/6J mice with acute Toxoplasma gondii infection and 8 normal healthy paired mice were prepared with WCX magnetic beads, and then analyzed on PBS II -C mass spectrometer reader. The protein spectra of the serum samples were normalized by the Ciphergen Protein Chip software. The peak labeling was performed by the Biomarker Wizard software. The specific protein biomarkers were screened by the Biomarker Pattern software to construct a diagnostic model for acute Toxoplasma gondii infection. RESULTS: A total of 13 distinguished proteomic peaks were detected. Nine peaks were of up-regulated expressions including m/z values of 1 932.76, 1 976.85, 2 090.53, 5 004.5, 5 776.01, 5 803.05, 5 847.99, 5 877.51 and 7 501.58, respectively; and four peaks were of down-regulated expressions including m/z values of 1 866.40,4 063.71, 8 120.31 and 8 203.83, respectively. CONCLUSION: The potential protein biomarkers for acute Toxoplasma gondii infection are discovered in mouse serum by MALDI-TOF-MS combined with WCX magnetic beads.

[Expression of Toll-like receptor 4 in brain tissue of chronic Toxoplasma gondii infection rats and its effect on brain injury].

OBJECTIVE: To explore the expression of Toll-like receptor 4 (TLR4) in brain tissue of chronic Toxoplasma infection rats and its effect on brain injury. METHODS: Ten male SD rats were randomly divided into 2 groups, namely control and infection groups. Each rat in the infection group was intraperitoneal injected with Toxoplasma gondii tachyzoites 10(7)/ml x 2 ml, and that in the control group was injected with 2 ml sterile normal sodium. After 10 weeks, the expression of TLR4 mRNA in the brain was determined by RT-PCR, and the levels of IL-1beta and IL-4 in peripheral blood sera were detected by ELISA. RESULTS: Compared with the control group, the expression of TLR4 gene and the peripheral blood serum level of IL-1beta of rats in the Toxoplasma gondii infection group were both significantly increased, with all P values were less than 0.05, and the level of IL-4 was also increased, but the difference had no statistically significance (P > 0.05). CONCLUSION: TLR4 might be involved in inflammatory reactions of brain injury for chronic Toxoplasma gondii infection rats.

[Effect of latent asymptomatic toxoplasmosis on glucose metabolism in brain of mice].

OBJECTIVE: To explore the effect of latent asymptomatic Toxoplasma gondii infection on glucose metabolism in brain of mice. METHODS: Twenty mice were randomly divided into two groups: a Toxoplasma infected group and normal control group. The mice in the Toxoplasma infected group were inoculated with 0.3 ml of brain suspension in saline containing ten Toxoplasma gondii tissue cysts, avirulent Toxoplasma gondii Prugniaud (PRU, a Type II strain). The mice in the control group received 0.3 ml of saline orally. Six monthes after the infection, the glucose metabolism changes in the mouse brain were evaluated by MicroPET, then all the mice were sacrificed and the brain tissues were observed histopathologically. RESULTS: Compared with the normal controls, the infected mice demonstrated profound and widespread brain pathology, and MicroPET indicated a significant glucose metabolism reduction in the brain of asymptomatic Toxoplasma gondii infected mice. CONCLUSION: Chronic Toxoplasma gondii infection maybe results in the glucose metabolism reduction in the brain of mice.