Comparative histopathology of mice infected with the 17XL and 17XNL strains of Plasmodium yoelii.

Plasmodium yoelii 17XL was used to investigate the mechanism of Plasmodium falciparum-caused cerebral malaria, although its histological effect on other mouse organs is still unclear. Here, histological examination was performed on mice infected with P. yoelii 17XL; the effect of P. yoelii 17XL infection on anemia and body weight loss, as well as its lesions in the brain, liver, kidney, lung, and spleen, also was investigated. Plasmodium yoelii 17XL-infected red blood cells were sequestered in the microcirculation of the brain and in the kidney. Compared with the nonlethal P. yoelii 17XNL strain, infection by P. yoelii 17XL caused substantial pulmonary edema, severe anemia, and significant body weight loss. Although P. yoelii 17XNL and 17XL produced a similar focal necrosis in the mouse liver, infection of P. yoelii 17XL induced coalescing of red and white pulp. Mortality caused by P. yoelii 17XL may be due to cerebral malaria, as well as respiratory distress syndrome and severe anemia. Plasmodium yoelii 17XL-infected rodent malaria seems to be a useful model for investigating severe malaria caused by P. falciparum.

Bone marrow mesenchymal stem cells can be mobilized into peripheral blood by G-CSF in vivo and integrate into traumatically injured cerebral tissue.

The efficacy of granulocyte colony-stimulating factor (G-CSF) in mobilizing mesenchymal stem cells (MSCs) into peripheral blood (PB) and the ability of PB-MSCs incorporated into injured brain were tested. Colony forming, cell phenotype and differentiation potential of mouse MSCs mobilized by G-CSF (40 µg/kg) were evaluated. Mortality and pathological changes in mice with serious craniocerebral trauma plus G-CSF treatment (40 µg/kg) were investigated. Bone marrow (BM) cells derived from GFP mice were fractionated into MSCs, hematopoietic stem cells (HSCs), and non-MSC/HSCs using magnetic beads and adherent culture. The resultant cell populations were transplanted into injured mice. The in vivo integration and differentiation of the transplanted cells were detected immunocytochemically. The expression of SDF-1 in injured area of brain was tested by Western blot. G-CSF was able to mobilize MSCs into PB (fourfold increase). PB-MSCs possessed similar characteristics as BM-MSCs in terms of colony formation, the expression pattern of CD73, 44, 90, 106, 31 and 45, and multipotential of differentiation. Accumulative total mice mortality was lower in TG group (5/14) than that in T group (7/14). It was MSCs, not HSCs or non-MSC/HSC cells integrated into the damaged cerebral tissue and differentiated into cells expressing neural markers. Increased SDF-1 expression in injured area of brain was confirmed, which could facilitate the homing of MSCs to brain. G-CSF can mobilize MSCs into PB and MSCs in PB can integrate into injured cerebral tissue and transdifferentiated into neural cells and may benefit the repair of trauma.

Plasmodium yoelii: correlation of TEP1 with mosquito melanization induced by nitroquine.

The antimalarial drug nitroquine is not only an effective antimalarial drug, it is also able to induce the melanization of Plasmodium species. However, the molecular mechanisms of the recognition reaction induced by this drug remain unclear. Silencing of thioester-containing protein-1 (TEP1) significantly compromised the ability of Anopheles gambiae to melanize the Plasmodium, leading to investigation of the involvement of A. stephensi TEP1 in melanization induced by nitroquine. This study shows that (1) binding of AsTEP1 to oocysts, especially melanized oocysts, (2) after ingestion of anti-AsTEP1 antibody, the melanization rate in antibody-treated mosquitoes are significantly lower than in control mosquito (p<0.05). The results suggest that nitroquine is able to induce Plasmodium recognition by TEP1, possibly triggering the resulting melanotic encapsulation. Further elucidation of the molecular mechanisms of mosquito immunity induced by antimalarial drugs will provide theoretical evidence for the use of antimalarial drugs, and a meaningful pathway for the design of novel antimalarial drugs.

Involvement of prophenoloxidases in the suppression of Plasmodium yoelii development by Anopheles dirus.

Anopheles dirus is refractory to a rodent malaria parasite, Plasmodium yoelii, and melanized oocysts are manifested in infected mosquitoes. Prophenoloxidase (PPO) is a zymogen whose active form mediates melanotic encapsulation of invading pathogens in mosquitoes. In this study, we cloned cDNA fragments of four An. dirus PPOs, that are orthologs of Anopheles gambiae PPO2, PPO4, PPO5 and PPO6. AdPPO4 expression in hemocytes was induced in response to P. yoelii infection. RNA interference using double stranded RNA of AdPPO4 led to depletion of its mRNA and other PPO transcripts. This depletion increased P. yoelii infection prevalence and oocyst intensity, and abolished the melanization of oocysts as well. Therefore, An. dirus PPOs may play a role in the refractoriness to P. yoelii.

[Inhibition of CpG oligodeoxynucleotide on the development of pre-erythrocytic stage of Plasmodium].

OBJECTIVE: To study the role of cytidine-phosphate-guanosine oligodeoxynucleotide (CpG ODN) on the development of Plasmodium liver stage. METHODS: Plasmodium yoelii BY265 18S rRNA was cloned, and the TaqMan real-time PCR was established on P. yoelii BY265 18S rRNA and mouse GAPDH as quantitative analysis model. The model was tested by the level of liver Plasmodium load with the liver cDNA in BALB/c mice infected by salivary gland sporozoites for 42 hours. Twelve BALB/c mice were randomly divided into CpG group, CpG control group and PBS control group which were injected respectively by ODN1826 30 microg, ODN1826 control 30 microg and 0.01 mol/L PBS 200 microl via vena caudalis. Twenty-four hours later, each mouse was inoculated with 100 sporozoites. Mice were sacrificed in 42 hours after infection, and the liver load of Plasmodium was analyzed by TaqMan real-time PCR. RESULTS: The cloned Py BY265 18S rRNA gene showed 98% similarity to Py 17XNL. The quantitative analysis model consisted by 18S rRNA and GAPDH showed positive correlation between the level of liver Plasmodium load and the sporozoite inoculation dose to mice. The Plasmodium load in CpG ODN pre-treated mice was reduced to one fifth of the control group (0.28/1.33) (P<0.05). CONCLUSION: The quantitative analysis model of TaqMan RT-PCR can detect the liver load of Plasmodium parasites, and CpG ODN can inhibit the development of its pre-erythrocytic stage.

[Inhibition of Plasmodium yoelii circumsporozoite protein on the activation of nuclear transcription factor in hepatoma cells stimulated by TNF-alpha].

OBJECTIVE: To investigate on the effect of Plasmodium yoelii (BY265 strain) circumsporozoite protein (CSP) on the activation of nuclear transcription factor KB (NF-KB) in hepatoma cells (HepG2) stimulated by TNF-alpha. METHODS: Entire coding sequence of CSP was reverse transcribed and amplified by RT-PCR with sporozoite total RNA as template, then cloned into pFLAG-CMV8 for construction of the recombinant plasmid pFLAG-CMV8-CSP. Indirect immunofluorescence staining with rabbit anti-CSP was applied to verify the expression and distribution of FLAG-CSP fusion protein in HepG2. Three groups were established for the experiment: group A with HepG2 transfected by pFLAG-CMV8 as negative control, group B with HepG2 transfected by pFLAG-CMV8 and stimulated by 100 ng/ml TNF-alpha, and group C with HepG2 transfected by pFLAG-CMV8-CSP and stimulated by 100 ng/ml TNF-alpha. Dual-luciferase assay and EMSA were performed to detect the nuclear translocation and activation of NF-kappaB, to observe if pFLAG-CMV8-CSP suppressed the activation of NF-KB in HepG2 stimulated by TNF-alpha. RESULT: The expression of pFLAG-CMV8-CSP was localized on cytoplasm of HepG2. The activity ratio of firefly luciferase to Renilla luciferase in group C (0.228 +/- 0.029) was significantly lower than both groups A (0.438 +/- 0.085) and B (0.571 +/- 0.030) (P < 0.05). EMSA showed that the band in group C was significantly weaker than group B. CONCLUSION: Plasmodium yoelii CSP localizes in the cytoplasm of HepG2 and inhibits the activation and nuclear translocation of NF-kappaB in HepG2 stimulated by TNF-alpha.

[Recombinant Plasmodium yoelii expressing green fluorescent protein in erythrocytic and mosquito stages].

OBJECTIVE: To generate recombinant Plasmodium yoelii BY265 strain which can express green fluorescent protein (GFP) in erythrocytic and mosquito stages. METHODS: Recombinant plasmid containing P. berghei ssu-rrna and GFP genes were linearized by Sac II. The linearized plasmid was introduced into the erythrocytic stage of P. yoelii by electroporation. Kunming mice were infected with the recombinant parasites. After 24-30 hours post-infection, mice were treated with 70 microg/ml of pyrimethamine intraperitoneally for 5-6 d, and tail vein blood was then collected to make smear for parasite count. The parasites were examined by PCR. Anopheles stephensi mosquitoes were used to bite mice infected with the recombinant parasite. At the 7th and 16th day after the bite, oocyst development in mosquitoes was observed by fluorescence microscopy. RESULTS: The recombinant parasites expressed GFP in erythrocytic stage, and the GFP and ssu-rrna genes were amplified by PCR in the recombinant parasites. The mosquito infection experiment showed a normal development of the recombinant parasites. CONCLUSION: Transgenic P. yoelii BY265 strain has been established with stable expression of GFP in both erythrocytic and mosquito stages.