AIM: A method was developed to separate contaminant-free viable Toxoplasma gondii cysts from brain samples of infected mice for molecular biology studies and reinfection.
MATERIALS AND METHODS: The mice brains were homogenized and washed with phosphate buffered saline (PBS) Tween 80 prior to fractionation using 19-22% dextran solution. Finally, the supernatant was purified by two-step membrane filtration (100-160 microm and < 10 microm) to obtain pure T. gondii cyst. The isolates were analyzed through microscopic observation, qPCR and by reinfection of new batch of mice.
RESULTS: T. gondii cysts were best isolated with 21% dextran solution and two step filtration.
CONCLUSIONS: The method was observed not to disrupt the integrity of the cysts containing bradyzoites. In addition, the isolated cysts in the filtrate were found to be contaminant-free, viable and able to infect healthy mice when introduced orally; which, mimics the natural infectivity pathway.
Synthetic glucocorticoid (dexamethasone; DEX) treatment during the neonatal stage is known to affect reproductive activity. However, it is still unknown whether neonatal stress activates gonadotropin-inhibitory hormone (GnIH) synthesizing cells in the dorsomedial hypothalamus (DMH), which could have pronounced suppressive action on gonadotropin-releasing hormone (GnRH) neurons, leading to delayed pubertal onset. This study was designed to determine the effect of neonatal DEX (1.0mg/kg) exposure on reproductive maturation. Therefore, GnRH, GnIH and GnIH receptors, G-protein coupled receptors (GPR) 147 and GPR74 mRNA levels were measured using quantitative real-time PCR in female mice at postnatal (P) days 21, 30 and in estrus stage mice, aged between P45-50. DEX-treated females of P45-50 had delayed vaginal opening, and irregular estrus cycles and lower GnRH expression in the preoptic area (POA) when compared with age-matched controls. The expression levels of GPR147 and GPR74 mRNA in the POA increased significantly in DEX-treated female mice of P21 and P45-50 compared to controls. In addition, GPR147 and GPR74 mRNA expression was observed in laser captured single GnRH neurons in the POA. Although there was no difference in GnIH mRNA expression in the DMH, immunostained GnIH cell numbers in the DMH increased in DEX-treated females of P45-50 compared to controls. Taken together, the results show that the delayed pubertal onset could be due to the inhibition of GnRH gene expression after neonatal DEX treatment, which may be accounted for in part by the inhibitory signals from the up-regulated GnIH-GnIH receptor pathway to the POA.
AIM: To determine the usefulness of prednisolone in increasing the number of Toxoplasma (T.) gondii tachyzoites and bradyzoites in mice.
MATERIALS AND METHODS: The mice were water-fasted prior to being immunosuppressed with oral inoculation of prednisolone. Tachyzoites of 7T gondii RH strain were inoculated into mice and the number of the parasites in the intraperitoneal fluids was then determined at 96 hs post-infection. In addition, tachyzoites of T. gondii ME49 strains were orally introduced into mice and the number of brain cysts formed was observed by microscopic observation at 45 days post-infection.
RESULTS: T. gondii propagation was found to be significantly improved by introduction of the prednisolone (p = 0.0004); and the number of parasite showed positive correlation with the increment in dosage of prednisolone (r = 0.9051).
CONCLUSIONS: The use of prednisolone greatly improved the number of parasite formed in mice: both tachyzoite and cyst forms.
Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.
BACKGROUND: Pleurotus sajor-caju (P. sajor-caju) has been extremely useful in the prevention of diabetes mellitus due to its low fat and high soluble fiber content for thousands of years. Insulin resistance is a key component in the development of diabetes mellitus which is caused by inflammation. In this study, we aimed to investigate the in vivo efficacy of glucan-rich polysaccharide of P. sajor-caju (GE) against diabetes mellitus and inflammation in C57BL/6J mice fed a high-fat diet.
METHODS: Diabetes was induced in C57BL/6J mice by feeding a high-fat diet. The mice were randomly assigned to 7 groups (n=6 per group). The control groups in this study were ND (for normal diet) and HFD (for high-fat diet). The treated groups were ND240 (for normal diet) (240 mg/kg b.w) and HFD60, HFD120 and HFD240 (for high-fat), where the mice were administrated with three dosages of GE (60, 120, 240 mg GE/kg b.w respectively). Metformin (2 mg/kg b.w) served as positive control. The glucose tolerance test, glucose and insulin levels were measured at the end of 16 weeks. Expressions of genes for inflammatory markers, GLUT-4 and adiponectin in the adipose tissue of the mice were assessed. One-way ANOVA and Duncan's multiple range tests (DMRT) were used to determine the significant differences between groups.
RESULTS: GE treated groups improved the glucose tolerance, attenuated hyperglycemia and hyperinsulinemia in the mice by up-regulating the adiponectin and GLUT-4 gene expressions. The mice in GE treated groups did not develop insulin resistance. GE also down-regulated the expression of inflammatory markers (IL-6, TNF-α, SAA2, CRP and MCP-1) via attenuation of nuclear transcription factors (NF-κB).
CONCLUSION: Glucan-rich polysaccharide of P. sajor-caju can serve as a potential agent for prevention of glucose intolerance, insulin resistance and inflammation.
We have investigated the effect of three potential scar-reducing agents applied at a sciatic nerve repair site in C57-black-6 mice. Under anaesthesia the nerve was transected, repaired using four epineurial sutures, and 100 μl of either triamcinolone acetonide (1 mg/100 μl), an interleukin-10 peptide fragment (125 ng/100 μl or 500 ng/100 μl) or mannose-6-phosphate (M6P, 200 mM or 600 mM) was injected into and around the nerve. After 6 weeks the extent of regeneration was assessed electrophysiologically by determining the ratio of the compound action potential (CAP) modulus evoked by electrical stimulation of the nerve 2 mm distal or proximal to the repair site. The conduction velocity of the fastest components in the CAP was also calculated. The percentage area of collagen staining (PAS) at the repair site was analysed using Picrosirius Red and image analysis. Comparisons were made with a placebo group (100 μl of phosphate buffered saline) and sham-operated controls. The median CAP modulus ratio in the 600 mM M6P group was 0.44, which was significantly higher than in the placebo group (0.24, P=0.012: Kruskal-Wallis test). Conduction velocities were also faster in the 600 mM M6P group (median 30 m s(-1)) than in the placebo group (median 27.8 m s(-1); P=0.0197: Kruskal-Wallis test). None of the other treated groups were significantly different from the placebo, and all had significantly lower CAP ratios than the sham controls (P<0.05). All repair groups had a significantly higher PAS for collagen than sham controls. We conclude that the administration of 600 mM mannose-6-phosphate to a nerve repair site enhances axonal regeneration.
Citalopram is the most potent selective serotonin reuptake inhibitor (SSRI) which is used as an antidepressant but causes sexual dysfunction. Whether citalopram induced sexual dysfunction is a result of gonadotropin-releasing hormone (GnRH), kisspeptin or RF-amide related peptide (RFRP) alteration is unknown. In this study, we tested mice for sexual behavior after vehicle (0.9% NaCl) and citalopram treatment (5 mg/kg) daily for 1 day (acute) and 21 or 28 days (chronic). Effects of acute and chronic treatments on neuronal numbers and mRNA expression of GnRH, kisspeptin and RFRP were measured. In addition, RFRP fiber projections to preoptic (POA)-GnRH neurons were analyzed using double-label immunohistochemistry. The expression of 14 different serotonin receptor types mRNA was examined in immunostained laser dissected single RFRP neurons in the dorsomedial hypothalamus (DMH), however only 11 receptors types were identified. Acute citalopram treatment did not affect sexual behavior, whereas, the total duration of intromission was reduced with chronic treatment. There was no effect in the expression of kisspeptin (neuronal numbers and mRNA) in the anteroventral periventricular nucleus and the arcuate nucleus and expression of GnRH (neuronal numbers and mRNA) in the POA after citalopram treatment. However, RFRP neuronal numbers in the DMH and fiber projections to the POA were significantly increased after chronic citalopram treatment, which suggests citalopram induced inhibition of sexual behavior involves the modulation of RFRP through serotonin receptors in the DMH.
Anti-idiotype (Id) vaccine therapy has been tested and shown to be effective, in several animal models, for triggering the immune system to induce specific and protective immunity against bacterial, viral and parasitic infections. The administration of anti-Id antibodies as surrogate tumor-associated antigens (TAA) also represents another potential application of the concept of the Id network. Limited experience in human trials using anti-Id to stimulate immunity against tumors has shown promising results. In this "counter-point" article, we discuss our own findings showing the potential of anti-Id antibody vaccines to be novel therapeutic approaches to various human cancers and also discuss where anti-Id vaccines may perform better than traditional multiple-epitope antigen vaccines.
The intraspecific variation of four laboratory-reared isolates of Taenia taeniaformis the SRN and KRN isolates from Norway rats, Rattus norvegicus, captured in Japan and Malaysia, respectively; the BMM isolated from a house mouse, Mus musculus, captured in Belgium; and the ACR isolate from a gray red-backed vole, Clethrionomys rufocanus bedfordiae, captured in Japan was examined by various criteria. Eggs of each of the four isolates were orally inoculated into several species of intermediate host. They were most infective to the rodent species from which the original metacestode of each isolate had been isolated in the field, and only the ACR isolate was infective to the gray red-backed vole. Although little difference was found between the SRN, KRN, and BMM isolates by the other criteria, including the morphology of rostellar hooks, the protein composition of the metacestode, and restriction endonuclease analysis of DNA, the ACR isolate was clearly different from the others. It was considered that the ACR isolate was independent as a strain distinct from the other three isolates.
Mice were chronically infected with cysts of ME49 strain of Toxoplasma gondii. At different periods post-infection, their spleens were removed and single cell suspensions were made. Lymphocyte transformation experiments were performed on the lymphocyte suspensions using three different kinds of antigens of ME49 strain of T. gondii, namely soluble, excretory/secretory and cystic forms. The results showed that the pattern of lymphocyte responsiveness was dependent on the kind of antigen employed for induction of the blastogenesis. Using soluble and cystic forms of the antigen, different periods of lymphocyte suppression and lymphocyte proliferation were demonstrated. However, with the use of excretory/secretory antigen, no significant suppression of lymphocyte stimulation was noted throughout the course of infection. Thus excretory/secretory antigen may be the best form of antigen for stimulation of the cell-mediated immune response and hence it appears to be a good candidate for vaccine in toxoplasmosis.
C57BL/6 mice were orally infected with different doses of cysts of ME49 strain of Toxoplasma gondii to produce groups of acutely and chronically infected mice. Sera were obtained at different periods post-infection. SDS-PAGE was ran with excretory/secretory antigens of ME49 and RH strains of T. gondii, followed by Western blot analyses using the above sera and anti- IgA, IgM, IgG as conjugates. The SDS-PAGE profiles of the two antigens were similar. However the antigenic bands showed variations in all blots, most evidently in IgA blots of chronic sera. IgG blots showed greatest similarities in reactive bands. In IgM blots, more common bands were shown in chronic sera than in acute sera. Variations and similarities in prominence of some bands and time of their appearance were also noted, especially in IgM and IgG blots of chronic sera. Thus antigenic variations and similarities are present in excretory/secretory products of different strains of T. gondii.