Plasmodium circumsporozoite protein suppresses the growth of A549 cells via inhibiting nuclear transcription factor κB.

Blocking the activation of nuclear factor κB (NF-κB) is a promising strategy for the treatment of non-small cell lung cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was previously reported to block NF-κB activation in hepatocytes. Therefore, in the present study, the effect of CSP on the growth of the human lung cancer cell line, A549, was investigated. It was demonstrated that transfection with a recombinant plasmid expressing CSP was able to inhibit the proliferation of A549 cells in a dose-dependent manner and induce the apoptosis of A549 cells. A NF-κB gene reporter assay indicated that CSP and its nuclear localization signal (NLS) motif were able to equally suppress the activation of NF-κB following stimulation with human recombinant tumor necrosis factor (TNF)-α in A549 cells. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but was able to markedly inhibit the nuclear translocation of NF-κB in TNF-α stimulated A549 cells. Therefore, the data suggest that CSP may be investigated as a potential novel NF-κB inhibitor for the treatment of lung cancer.

Complete mitochondrial genomes of Anopheles stephensi and An. dirus and comparative evolutionary mitochondriomics of 50 mosquitoes.

To better understand the phylogeny and evolution of mosquitoes, the complete mitochondrial genome (mitogenome) of Anopheles stephensi and An. dirus were sequenced and annotated, and a total of 50 mosquito mitogenomes were comparatively analyzed. The complete mitogenome of An. stephensi and An. dirus is 1,5371 bp and 1,5406 bp long, respectively. The main features of the 50 mosquito mitogenomes are conservative: 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, positive AT-skew and negative GC-skew. The gene order trnA-trnR in ancestral insects is rearranged. All tRNA genes have the typical clover leaf secondary structure but tRNA Ser . The control regions are highly variable in size. PCGs show signals of purifying selection, but evidence for positive selection in ND2, ND4 and ND6 is found. Bayesian and Maximum Likelihood phylogenetic analyses based on all PCG nucleotides produce an identical tree topology and strongly support the monophyly of subgenera Cellia, Anopheles, Keterszia and Nyssorhynchus, the sister relationship of the subgenera Nyssorhynchus and Keterszia, and Cellia and Anopheles. The most recent ancestor of the genus Anopheles and Culicini + Aedini exited ~145 Mya ago. This is the first comprehensive study of mosquito mitogenomes, which are effective for mosquito phylogeny at various taxonomic levels.

[The effect of leptin transgenic Plasmodium yoelii on mouse body weight].

Objective: To investigate the effect of leptin transgenic Plasmodium yoelii on mouse body weight. Methods: To construct the leptin gene-containing CRISPR/Cas9 recombinant plasmid which had the 5'UTR and 3'UTR of MIF(macrophage migration inhibitory factor) of Plasmodium yoelli 17XNL strain at two ends, the exogenous mouse leptin gene was inserted downstream of MIF coding region through homologous recombination, resulting in the PYC-MIF-Leptin recombinant plasmid. The recombinant plasmid was then electroporated into P. y 17XNL mature schizonts, and the transgenic schizonts were used to infect a Kunming mouse via tail vein injection. The trangenic P. y clone was screened by pyrimethamine selection and identified by PCR. The trangenic or wild-type P. y was used to infect a C57BL/6 mouse respectively. Blood sample was collected through eye ball and tail vein, and immunofluorescence and RT-PCR were performed to determine the expression of leptin protein in the parasites. Finally, PBS (200 µl) containing trangenic or wild-type P. y (1 × 104) was injected through the tail vein into C57BL/6 mice(n = 5 respectively). The negative control received a same volume of PBS. The changes of parasitemia and body weight were recorded every two days. Results: The leptin-expressing recombinant plasmid PYC-MIF-Leptin was constructed successfully. Results of DNA sequencing of transgenic parasites confirmed the integration of leptin gene at the downstream of MIF gene and successful transcription. Immunofluorescence results indicated successful expression of mouse leptin protein. The weight loss was significant in mice infected with transgenic parasites on day 17(17.26 ± 1.40)g, decreased by 10.7%, but not in the other two groups. Both transgenic and wild-type parasites began to decline when parasitemia reached about 10%, but the transgenic parasites proliferated more rapidly. Both disappeared at 23 days. Conclusion: Infection with leptin transgenic parasites decreases the body weight of the infected mice.

[Establishment and evaluation of loop-mediated isothermal amplification based on Plasmodium falciparum unique genes coding PHIST proteins].

OBJECTIVE: To establish a novel convenient loop-mediated isothermal amplification (LAMP) method with the unique genes coding Plasmodium helical interspersed sub-telomeric superfamily (PHIST) for the rapid molecular diagnosis of P. falciparum. METHODS: The unique genes coding PHIST with high expression mRNA profile during the ring form or schizont period of P. falciparum were screened and selected from the PlasmoDB database. The LAMP primers of targeted genes were designed by the online software (PrimerExplorer V4). The LAMP assay was executed by the color-displaying method with SYBR Green. The dried blood spots of P. falciparum from clinical isolates were collected and the genomic DNA (gDNA) was extracted. For evaluation of sensitivity, the gDNA was diluted to four gradients (10⁻¹, 10⁻², 10⁻³, and 10⁻4). For assessment of specificity, the gDNA (s) of P. vivax, P. yoelii, Taenia saginata, and Schistosoma japonicum were also extracted. RESULTS: Totally, 61 P. falciparum unique genes coding PHIST were found. The PF3D7_1372300 with high expression value during the ring form and PF3D7_1401600 with high expression value during the schizont period were selected for LAMP assay. The lowest detectable limits of PF3D7_1372300 and PF3D7_1401600 were 130.5 parasite/µl and 1305.3 parasite/µL, respectively. Specific tests showed the amplified products of P. falciparum was positive and all the others including P. vivax, P. yoelii, T. saginata, and S. japonicum were negative. CONCLUSIONS: The established LAMP method with PF3D7_1372300 gene is sensitive, specific, simple and useful. It can be applied to the field investigation and clinical diagnosis for falciparum malaria.

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.

[Insect odorant receptors and their olfactory signal transduction pathway].

Protection against insect bites is one of the main strategies in prevention and control of the vector-borne diseases. However, due to the obvious shortcomings of traditional control methods, it is necessary to develop new control measures. Most insects rely on their olfactory systems for host and mate location. Interfering with insect olfactory systems is becoming a hot research area in the control of vector-borne diseases. As odorant receptors play a major role in perception of odorant molecules by insect olfactory system, this paper summarizes the recent progress on insect odorant receptors and their olfactory signal transduction.

[Immunopathological mechanism of cerebral malaria].

Cerebral malaria is a severe complication of malaria. Early studies suggest that cerebral malaria is related to cytoadherence of parasitized red blood cells to the microvessel endothelium of brain. However, more and more evidence supported that the cause of cerebral malaria is uncontrolled inflammatory cytokines and infiltration of lymphocytes in brain microvessel. The article summarizes the research progress on immunological mechanism of cerebral malaria.

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.

[Mechanism of protective immunity induced by irradiation-attenuated sporozoites and its implication for pre-erythrocytic malaria vaccine research].

Irradiation-attenuated sporozoites are still the most effective pre-erythrocytic malaria vaccine However, limitation of purified sporozoites and difficulty in attenuation controlling of sporozoites hamper its use in practice. Understanding the mechanism of protective immunity induced by irradiation-attenuated sporozoites will be helpful for the design of the efficient pre-erythrocytic malaria vaccine. This is a review on research progress in the mechanism of protective immunity induced by irradiation-attenuated sporozoites and current status of pre-erythrocytic malaria vaccine development.

[Correlation of Anopheles TEP1 gene with melanization induced by nitroquine].

OBJECTIVE: To analyze the relationship between the TEP1 gene of Anopheles stephensi and melanotic encapsulation of Plasmodium yoelii induced by anti-malaria drug nitroquine. METHODS: Haemolymph samples from three groups of An. stephensi fed with sucrose solution, Plasmodium-infected blood and nitroquine, respectively, were collected at the 1st, 2nd, 3rd and 4th day after drug administration. Degenerate primers were designed according to the conserved amino acid sequence within TEPs of the mosquitoes. Fluorescent quantitation PCR was used to detect the variation of TEP1 gene transcript induced by nitroquine. The melanization of oocysts was observed by light microscopy. RESULTS: TEP1 gene was cloned, the predicted amino acid residues harbored a highly conserved canonical thioester motif GCGEQ. The fluorescent quantitation PCR revealed that nitroquine induced an up-regulation of TEP1 activity. The transcription of TEP1 gene in nitroquine treated group (2.423) was significantly higher than that of the infected blood-fed group(1.036) at the 3rd day after nitroquine treatment (P<0.05). At the same time, most oocysts were found to be encapsulated in nitroquine treated group, while no melanized parasites were observed in the infected blood-fed group. CONCLUSION: Transcriptional variation of TEP1 gene may be related to the melanization induced by nitroquine.

[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.

[IFN-gamma promotes human monocytes to differentiate to untypical mature DC].

AIM: To explore the morphologic and phenotypic changes of monocytess in response to the stimulation of IFN-gamma, TNF-alpha and IFN-alpha. METHODS: Newly isolated monocytes from human PBMCs were treated with IFN-gamma, TNF-alpha or IFN-alpha. The phenotypic growth of monocytes with or without stimulation was observed by microscope. The surface expression of CD1a, CD14, CD80, CD83, CD86, and HLA-DR on the monocytes was assayed by flow cytometry. RESULTS: After IFN-gamma stimulation, monocytes displayed the shape of fusiform or polygon and were more likely to adhere and cluster to form the distinct structure as "cell islet". IFN-gamma; also induced or up-regulated the surface expression of CD80, CD83, CD86, and HLA-DR on the monocytes and down-regulated the CD14 expression. IFN-gamma even induced the CD1a expression and changed the monocytes phenotype from CD14(+)CD1a(-)CD83(-) to CD14(+)CD1a(+)CD83(+) after stimulated for 5 days. Cytokines TNF-alpha and IFN-alpha; have the different impacts on monocytes from IFN-gamma. CONCLUSION: IFN-gamma have the immunoregulatory function of making monocytes differentiate to untypical mature dendritic cells (DC).

Plasmodium yoelii: correlation of up-regulated prophenoloxidase and phenoloxidases with melanization induced by the antimalarial, nitroquine.

Although knowledge of the mosquito immune response has recently improved, less is known about the impact of antimalarial drugs on mosquito immunity. In the present study, we found that nitroquine, an effective antimalaria drug, could also induce melanotic encapsulation of Plasmodium by Anopheles stephensi. The melanization rate of the nitroquine treated group was 60.8%. To explore the effect of nitroquine on mosquito immunity, we determined the increase in activity of phenoloxidases (PO) enzyme, the main component of melanotic encapsulation, with nitroquine treatment. Moreover, we cloned prophenoloxidase (PPO) gene, which is accepted as the inactive phenoloxidase form and observed inducible expression of this gene with nitroquine treatment by real-time PCR. Our data implied that up-regulation of PPO gene and PO activity might be correlated with nitroquine. Nevertheless, nitroquine had no effect on the transcription of PPO gene or the activity of PO enzyme in the mosquito fed on a normal blood meal. In our study, we also observed the degenerative effect of 0.1% nitroquine on Plasmodium in the mosquito. This suggests that the degeneration of Plasmodium induced by nitroquine might result in the exposure of pattern-recognition ligands which can active the immune reaction, up-regulate PPO gene expression and PO activity, and induce the melanization.

TLR2 and TLR4 agonists synergistically up-regulate SR-A in RAW264.7 through p38.

It is known that macrophage scavenger receptor A (SR-A) can protect mice from endotoxemia. In addition, Escherichia coli O111:B4 LPS from Sigma (sLPS), which contains both TLR4 and TLR2 agonists, was previously reported to be able to induce SR-A expression on murine macrophage cell line RAW264.7. However, the relative role of both TLR4 and TLR2 agonists from Sigma (sLPS) in the up-regulation of SR-A on RAW264.7 is still undefined. Here, we found that sLPS could only slightly up-regulate SR-A on RAW264.7 following removing its TLR4 and TLR2 agonists, respectively. In contrast, the combination of TLR4 agonist uLPS (re-extracted sLPS) and TLR2 agonist Pam3CSK4 dramatically induced SR-A expression, and synergistically promoted RAW264.7 to bind and internalize FITC-LPS specifically through SR-A. The combination had no such effect either on TLR2 or TLR4 expression, and incubation with IL-6, IL-10, IL-12 or TNF-alpha alone could not induce SR-A expression on RAW264.7. In addition, treatment with a NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) could only weakly suppress the up-regulation of SR-A by the combination. However, the combination synergistically promoted MAPK p38 phosphorylation, and p38 specific inhibitor SB203580 completely suppressed its inducible effect on SR-A expression. Hence, we demonstrated that up-regulation of SR-A by sLPS was resulted from the cooperation of its TLR4 and TLR2 agonists through p38, and we also presented a novel synergy effect of TLR2 and TLR4 agonists.

[Study on the relationship between intracellular free calcium and melanization in oocysts of Plasmodium yoelii].

OBJECTIVE: To study the change of intracellular free Ca2+ in the oocyst when it melanized and to find out the relationship between the melanized oocyst and its intracellular level of free Ca2+ in a Plasmodium-refractory strain of Anopheles dirus. METHODS: The distribution and experimental condition of the intracellular free Ca2+ in oocyst of Plasmodium yoelii was measured with Ca2+ sensitive dye Fluo-3/AM and Pluronic F-127 under confocal laser scanning microscope (CLSM) at different time. RESULTS: The best load condition was that the oocysts were incubated in 3 mumol/ml Fluo-3/AM adding 1 microliter/ml 25% Pluronic F-127 for 60 min at 37 degrees C. Fluorescent imaging of oocysts was affected by an increase or decrease of the concentration of Fluo-3/Am and incubation time. The distribution of intracellular free Ca2+ was heterogeneous in the oocysts. The mean value of Ca2+ in the mature oocysts was (137.15 +/- 7.02) nmol/L (X +/- S) but was (18.44 +/- 1.75) nmol/L in melanized oocysts with Ca2+ sedimentation in the wall of oocyst. CONCLUSION: The results suggest that the level of the intracellular free Ca2+ in oocyst decreased and excreted during its melanization in a Plasmodium-refractory anopheline mosquito species.

[The internal control role of ribosomal protein S7 in the defense of Anopheles dirus against Plasmodium infection].

OBJECTIVE: To investigate the role of ribosomal protein S7 (rpS7) in the defense of Anopheles dirus against infection. METHODS: rpS7 was amplified from Anopheles dirus hemocytes with degenerated primers designed according to the conservative region of S7, rpS7 was then cloned using T/A cloning kit and the inserted fragment was sequenced. The difference of the transcript abundance of rpS7 from Anopheles dirus hemocyte among non-blood-fed (N), normal-blood-fed (B) and Plasmodium yoelii infected groups (I) was also analyzed by RT-PCR and gel scanning system at d1, d2, d3, d4, d7 and d11 after blood feeding. RESULTS: There is no significant difference of rpS7 signal between the three groups. CONCLUSION: Anopheles dirus S7 can be used as an internal control to study the role of Anopheles dirus related immune factors in Plasmodium infection.