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Objective To learn whether plasmodium genetic attenuated sporozoites (GAS) can induce immunity against lung cancer ,in order to provide new ideas for the study of lung cancer vaccine .Methods Ther study was divided into two groups respec‐tively ,experimental group received intravenous injection of genetically attenuated sporozoites to immunize C57BL/6J mice and con‐trol group injection of phosphate buffer solution (PBS);after 14 days ,we subcutaneously inoculated lewis lung cancer (LLC) cells , calipers was used to measure tumor size .Immunohistochemical staining was detected tumor proliferation ,apoptosis ,and angiogene‐sis .Results There was statistically significant in tumor size .Immunohistochemical staining revealed that attenuated sporozoites in‐fection inhibited LLC eslls proliferation ,angiogenesis ,apoptosis .Conclusion The malaria attenuated sporozoites may provide a no‐vel strategy or therapeutic vaccine vector for anti‐lung cancer immune‐based therapy .
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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 de?signed 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 lim?its of PF3D7_1372300 and PF3D7_1401600 were 130.5 parasite/μl and 1 305.3 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. japoni?cum 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.
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Objective To investigate brain function alteration in early onset schizophrenia by amplitude of low-frequency fluctuation (ALFF) based on resting-state functional MRI (fMRI). Method Twenty patients with early on?set schizophrenia and 20 sex, age, education years matched healthy controls underwent resting-state functional MRI. The difference in ALFF was compared between patients and controls. Result Compared with control group, the ALFF values in patient group increased in the left frontal gyrus, precuneus, cingulate cortex, occipital lobe, parahippocampal gyrus and calcarine (P<0.05, AlphaSim revised), while decreased in the right superior temporal lobe and the posterior lobe of cerebellum (P<0.05, AlphaSim revised). Conclusion There are abnormal ALFF in multiple regions in early onset schizo?phrenia, suggesting that early onset schizophrenia may have altered in resting-state brain activity.
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BACKGROUND:It is reported that tailless-like protein (TLX) plays critical roles in the regulation of early developmental processes in vertebrates, and it plays a key role in stem cells proliferation and differentiation into neurons. OBJECTIVE: To construct recombinant plasmid pEGFPN1-TLX and study the transfection into dermal multipotential stem cells. DESIGN, TIME AND SETTING: Cytogene experiment was performed at the Department of Pathogen Biology, School of Basic Medical Science, the Third Military Medical University of Chinese PLA from March to December 2007. MATERIALS: An adult SD was obtained from the Experimental Animal Center of the Third Military Medical University of Chinese PLA; dermal moltipotential stem cells (DMSCs) were cultured by the Institute of Combined Injury of the Third Military Medical University of Chinese PLA; pEGFPN1 and DH5α was gifted by professor Xu.METHODS: Total RNA was extracted from rat brain tissue to amplify TLX-coded cDNA sequence using RT-PCR. T/A was cloned on pMD18-T vector and determined using BamHI and Hindlll. The products were positive recombinant plasmid pMD18-T-TLX segments, which were sub-cloned in pEGFPN1 to construct recombinant plasmid pEGFPN1-TLX. Finally, pEGFPN1-TLX was transfected into DMSCs.MAIN OUTCOME MEASURES: The fluorescence protein expression was observed under fluorescence microscope at 24 hours after transfection; TLX mRNA expression was detected using RT-PCR; neuronal differentiation was observed using immunohistochemical staining.RESULTS: TLX full length cDNA was successfully cloned into pEGFPN1, and pEGFPN1-TLX was successfully constructed by means of sequence analysis and enzyme cutting identification. As compared with non-transfected DMSCs, pEGFPN1-TLX transfected DMSCs were observed after 10 days, formed resistant clones after 15 days, and shown a green fluorescent protein expression. However, non-transfected DMSCs died at day 10. RT-PCR indicated that pEGFPN1-TLX transfected DMSCs could express TLX mRNA. At day 3 after induction, NF200 positive cells were increased, but glial fibrillary acidic protein positive cells were decreased after induction of pEGFPN1-TLX transfected DMSCs.CONCLUSION: TLX was successfully constructed and transfected into DMSCs. After transfection, neuronal differentiation of DMSCs was enhanced, and the differentiation to gliocytes was inhibited.
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Objective To construct cDNA library from adults of Anopheles dirus for cloning the immune genes or related genes for malaria parasites development. Methods The mRNA of adult Anopheles dirus was isolated. The library was constructed by using the Zap Express vector(Stratagene) and the quality was evaluated. Results The efficiency of the library was 2.1?10 6 pfu/ml with 98% clones positive. The average length of the insert fragment was over 1 kb. Conclusion cDNA library of adult Anopheles dirus with high efficiency can be constructed by using the Zap Express library construction Kit (Stratagene).
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Objective To observe the effect of blood feeding and plasmodium yoelii infection on the transcript abundance of ribosomal protein S7 from Anopheles dirus hemocyte Methods Anopheles dirus of 3～5 days old were divided into normal group (N), blood feeding group(B) and Plasmodium yoelii infection group(I) Hemocytes of 50 Anopheles dirus from each group were collected by centrifuge method on days 1, 2, 3, 4, 7 and 11 after blood feeding, respectively Then, all hemocyte samples were used for total RNA isolation and analyzed by RT PCR Finally, the same volume(10 ?l) of all the PCR products from each group was used for agarose gel electrophoresis and the data obtained were analyzed statistically Results There was no significant difference in ribosomal protein S7 signal between the three groups Conclusion Similar to Anopheles gambiae and human rpS7, Anopheles dirus rpS7 might be also used as an internal control for the studies of the role of Anopheles dirus related immune factors in attacking Plasmodium infection
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Objective To explore the relationship between hemolymph phenol oxidase and the melanization of Plasmodium yoelii oocysts in Anopheles dirus. Methods An Anopheles dirus-Plasmodium yoelii system was used Anopheles dirus were divided into 3 groups, that is, non-blood-fedding (N), normal-blood-fedding (B) and infected-blood-fedding (I). The activities of MPO and o-DPO in hemolymph from 3 groups were determined with native polyacrylamide gel electrophoresis (PAGE) and density scanning at 5, 7, 11 and 15 d after blood feeding. Results Both MPO and o-DPO activity were significantly higher in group I than group N and B (P<0.05). But with the melanization of Plasmodium yoelii oocysts, both MPO and o-DPO activity in group I were decreased in comparison with group N, especially on the 15 th day after infected-blood feeding. MPO and o-DPO activity in group B were significantly stronger than those of group N. Conclusion Blood feeding and infection of Plasmodium yoelii both can activate the cascade. The heamolymph phenol oxidase may play an important role in the melanization of Plasmodium yoelii oocysts in Anopheles dirus.
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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.
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Objective To clone the partial cDNA sequence of prophenoloxidase (PPO) gene of Anopheles dirus . Methods Degenerative primers were designed according to the conserved sequence blocks within the prophenoloxidase of insects. RNA sequence of the larva of Anopheles dirus was amplified by RT PCR to get the prophenoloxidase cDNA which was then cloned into T vector and sequenced. The partial cDNA sequence of prophenoloxidase gene was analysed and compared with other prophenoloxidase gene of insects. Results The partial cDNA sequence of AdPPO4 was 597 bp, and its deduced amino acid sequence was 199aa. The cDNA sequence homology and amino acid sequence homology was 84% and 90%, respectively, as compared with the PPO4 gene of Anopheles gambiae . Conclusion The AdPPO4, with high sequence homology with the PPO4 gene of Anopheles gambiae , is successfully cloned from the larva of Anopheles dirus .
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Objective To investigate the expression patterns of serine proteases Adsp1 and Adsp3 in the main tissue of Anopheles dirus and the effects of blood feeding and Plasmodium infection on the transcript abundance of Adsp1 and Adsp3 from Anopheles dirus haemocytes. Methods Haemocytes were collected by centrifugation. The midguts and salivary glands were dissected from 50 adult female Anopheles dirus of 3-5 d old for the extraction of total RNA for amplification by RT PCR. Anopheles dirus of 3-5 d old were divided into normal group (N), blood feeding group (B), and Plasmodium yoelii infection group (I). Haemocytes of 50 adult female Anopheles dirus from each group after blood feeding were also collected, and the total RNA was isolated at 1, 2, 3, 4, 7, and 11 d, respectively. Then, the same volume (10 ?l) of total RNA was analyzed by semi quantitative RT PCR with Ads7 as the internal control. Agarose gel analysis was performed on PCR products from each group. Results Expression of both Adsp1 and Adsp3 mRNA was found in the haemocytes and salivary glands, but not in the midguts. Semi quantitative RT PCR indicated that transcript abundance of AdsP1 and AdsP3 from Anopheles dirus haemocytes was enhanced after blood feeding and Plasmodium yoelii infection, especially during melanotic encapsulation of Plasmodium yoelii . Conclusion AdsP1 and AdsP3 may be involved in the melanotic encapsulation response of Plasmodium yoelii by Anopheles dirus , and may be the prophenoloxidase activating enzyme of Anopheles dirus .
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Objective To investigate the relationship between the immune defence reaction against Plasmodium infection and the prophenoloxidase (PPO) of the midgut by comparative analysis of the distributions and the changes of PPO in the midgut of Anopheles stephensi and Anopheles dirus before and after infection with Plasmodium yoelii . Methods Midguts were dissected out from both Anopheles stephensi and Anopheles dirus at 3, 5, 7, 11 and 15 d before and after infection with Plasmodium yoelii . Immunohistochemistry and Western blotting were performed respectively on the collected midguts using Manduca Sexta PPO IgG polyantibody. Results PPO in the midguts from both Anopheles stephensi and Anopheles dirus was mainly located in the circulation conduit of midgut before infection with Plasmodium yoelii , but aggregated and distributed at the interspace of midguts as pieced or/and stripped forms after infection. Furthermore, PPO in the midgut of Anopheles dirus was more concentrated than that of Anopheles stephensi . Western blotting revealed that the PPO band with about molecular weight of 67?10 3 was detected in the midguts of both Anopheles stephensi and Anopheles dirus before and after Plasmodium yoelii infection. There was significant difference before and after infection, and the PPO band was obviously enhanced after infection. PPO bands in the midgut of Anopheles dirus were more prominent than those of Anopheles stephensi . Conclusion PPO in the midgut of Anopheles mosquitoes may come from the hemolymph by the circulation conduit before Plasmodium yoelii infection. However, the different distributions and changes of PPO in the midguts resulted from the Anopheles mosquitoes infected with Plasmodia may be closely correlated with Plasmodia infection, which may be of important physiological significance and may be involved in the immune defensive reaction against Plasmodium .
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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 ?g,ODN1826 control 30 ?g and 0.01 mol/L PBS 200 ?l 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
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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 adminstration. 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 microcopy. 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
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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.
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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 Plutonic F-127 under confocal laser scanning microscope (CLSM) at different time. Results The best load condition was that the oocysts were incubated in 3 ?mol/ml Fluo-3/AM adding 1 ?l/ml 25% Pluronic F-127 for 60 min at 37 ℃ . 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-reiractory anopheline mosquito species.