MicroR-146 blocks the activation of M1 macrophage by targeting signal transducer and activator of transcription 1 in hepatic schistosomiasis.

Schistosomiasis is a chronic disease caused by the parasite of the Schistosoma genus and is characterized by egg-induced hepatic granulomas and fibrosis. Macrophages play a central role in schistosomiasis with several studies highlighting their differentiation into M2 cells involved in the survival of infected mice through limitation of immunopathology. However, little is known regarding the mechanisms of regulating macrophage differentiation. Here, we showed that the early stage of infection by Schistosoma japonicum induced expression of type 1T-helper-cell (Th1) cytokine, interferon-γ (IFN-γ), leading to increase in M1 cells. However, the presence of liver-trapped eggs induced the expression of Th2 cytokines including interleukin-4 (IL-4), IL-10, and IL-13 that upregulated the transcription of miR-146b by activating signal transducer and activator of transcription 3/6 (STAT3/6) that bind to the promoter of the pre-miR-146b gene. We found that the miR-146a/b was significantly upregulated in macrophages during the progression of hepatic schistosomiasis. The elevated miR-146a/b inhibited the IFN-γ-induced differentiation of macrophages to M1 cells through targeting STAT1. Our data indicate the protective roles of miR-146a/b in hepatic schistosomiasis through regulating the differentiation of macrophages into M2 cells.

[Research Progress on Artemisinin Resistance in Plasmodium falciparum].

Artemisinin (ART) is a novel and effective antimalarial drug discovered in China. As recommended by the World Health Organization, the ART-based combination therapies (ACTs) have become the first-line drugs for the treatment of falciparum malaria. ART and its derivatives have contributed greatly to the effective control of malaria globally, leading to yearly decrease of malaria morbidity and mortality. However, there have recently been several reports on the resistance of Plasmodium falciparum to ART in Southeast Asia. This is deemed a serious threat to the global malaria control programs. In this paper, we reviewed recent research progress on ART resistance to P. falciparum, including new tools for resistance measurement, resistance-associated molecular markers, and the origin and spread of the ART-resistant parasite strains.

[In vivo identification of the interaction between var intron and an ApiAP2 transcription factor in Plasmodium falciparum].

OBJECTIVE: To investigate the potential interaction between the ApiAP2 protein family member, PF3D7_1107800, and var intron of Plasmodium falciparum in vivo. METHODS: Genomic DNA was extracted from Plasmodium falciparum (3D7 strain), 5' end gene fragment of PF3D7_1107800 was amplified by PCR, and cloned into pGEX-4T-1 vector. The constructed pGEX-4T-1-PF3D7_1107800N was transformed into E. Coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was purified through glutathione sepharose. Twenty female BALB/c mice were divided into 2 group. Ten mice in experiment group were immunized with a mixture of the purified protein and Freund's adjuvant by subcutaneous injection. Other 10 mice received PBS injection as control. Sera from mice of 2 group were purified with protein G. The effect of the antibody was testified with Western blotting. DNA products of ChIP assay was analyzed for enrichment of anti-PF3D7_1107800 group in ups C var intron by qPCR. RESULTS: PCR result of the PF3D7_1107800 gene 5' end segment showed that there was a specific band (about 345 bp), which was consistent with the theoretical value. The constructed vector pGEX-4T-1-PF3D7_1107800N was confirmed by gene sequencing. SDS-PAGE and Western blotting analysis demonstrated that the recombinant protein was about Mr 37,000. The anti-PF3D7_1107800 serum was obtained after the immunization of mice with the purified protein, and reacted with the recombinant protein, the specific band was about Mr 200,000. qPCR result showed that the fold enrichment of anti-PF3D7_1107800 group in var intron was two times higher than that of the reference gene region. CONCLUSION: The Plasmodium falciparum ApiAP2 family member PF3D7_1107800 binds to ups C var intron region in vivo.

Analysis of var genes cloned from a Plasmodium falciparum isolate in China.

OBJECTIVE: To analyse the var gene repertoire and characterise the chondroitin sulphate A (CSA)-binding activity of the Duffy-binding like (DBL) domains encoded by the var2csa gene of a Plasmodium falciparum (P. falciparum) isolate in Hainan Province, China. METHODS: The sequences of var DBL1 regions were PCR-amplified, sequenced and the sequence characteristics was bioinformatically analysed. Recombinant proteins encoded by the var2csa genes were expressed and purified. The binding activities of the recombinant proteins to CSA receptor was detected by ELISA assays. RESULTS: Fifty six unique DBL α sequences were obtained, and the sequences represented similar diversity to the var genes of the genome parasite 3D7. There are two var2csa genes in the P. falciparum isolated from Hainan Province. Unlike in other falciparum parasites such as HB3, the two var2csa genes are more diverged. The receptor-binding capacity of DBL-5ε and DBL-6ε domains of HN var2CSA was studied. CONCLUSIONS: This work represented the diversity of var genes of a P. falciparum isolate in China.

[Research and development of malaria vaccine in China].

It remains an urgent need to develop effective malaria vaccine for global control of malaria. Application of high technologies such as biotechnology has facilitated the process of vaccine development for malaria. In the past 30 years, a large number of vaccine candidate antigens for malaria have been identified and some of them are currently in clinical trials. Major progress in malaria vaccine development has also been made in China. The PfCP-2.9 blood stage vaccine for malaria has entered clinical studies and some other vaccine candidates including combination malaria vaccine are currently in pre-clinical studies. The availability of various national research programs and international funding has stimulated laboratory and pre-clinical studies of malaria vaccine candidates. It remains a long-term goal to develop a safe and effective malaria vaccine to control and even eliminate the disease in the world, and many issues including malaria immunology and various types of technologies need to be addressed. However, efforts need to be continued toward the goal.

[Construction and expression of Plasmodium berghei chimeric protein in Pichia pastoris and its immunogenicity in mice].

OBJECTIVE: To produce an erythrocytic stage chimeric protein of Plasmodium berghei in Pichia pastoris and evaluate its immunogenicity. METHODS: The DNA sequences of AMA1 (III) and MSP1-19 from P. berghei homologous to the corresponding sequences of P. falciparum chimeric antigen 2.9 (PfCP-2.9) were fused to generate a chimeric gene, designated as PbCP-2.9. The resulting gene was redesigned using Pichia preferential coden usage and expressed in P. pastoris in the secreted form. The recombinant protein was purified by Ni-NTA affinity chromatography. Three groups each with 10 BALB/c mice were immunized subcutaneously with 20 microg of purified PbCP-2.9 antigen formulated in Freund's adjuvant, Montanide ISA720 and Montanide IMS 1312, respectively. Three control groups each with 10 mice received only adjuvants emulsified with PBS. All the mice received three immunizations at 2-week intervals with the same dose of antigen. Serum samples were collected at pre-immunization and one week after each immunization, and were analyzed for specific antibodies by ELISA and reaction with natural P. berghei proteins by IFAT. RESULTS: The PbCP-2.9 antigen with Mr 26400 was successfully expressed in P. pastoris in secreted form. The recombinant protein can be recognized by the serum against blood stage parasites of P. berghei. High antibody responses were detected in all three PbCP-2.9-immune groups of mice by ELISA. However, mice immunized with PbCP-2.9 antigen in Freund's adjuvant produced higher antibody titers than those with PbCP-2.9 antigen in Montanide ISA 206 and Montanide IMS 1312 adjuvants. The mean antibody titer in Freund's adjuvant was 6.9-fold higher than in Montanide ISA 206 adjuvant and 5.6-fold higher than in Montanide IMS 1312 adjuvant after the second immunization (F=81.06, P< 0.01). In addition, after the third immunization the mean antibody titer in Freund's adjuvant was 3.7-fold higher than in Montanide ISA 206 adjuvant and 3.3-fold higher than in Montanide IMS 1312 adjuvant (F=13.29, P< 0.01). The results from IFAT assay demonstrated that the immune sera recognized the surface proteins of P. berghei parasites. CONCLUSION: The coden-optimized PbCP-2.9 gene has been constructed and expressed in P. pastoris. The chimeric antigen is highly immunogenic in mice and the immune sera can interact with natural proteins of P. berghei parasite.

[Genotyping and polymorphism analysis of Plasmodium falciparum isolates by nest-PCR and PCR-RFLP].

OBJECTIVE: To analyze the efficiency and specificity of MSP2 alleles genotyping for Plasmodium falciparum isolates by Nest-PCR and PCR-RFLP. METHODS: MSP2 alleles from Plasmodium falciparum isolates of Yunnan and Hainan were genotyped by Nest-PCR and PCR-RFLP, respectively, and the efficiency and specificity of the two methods were analyzed. RESULTS: The conventional Nest-PCR method could detect 79.8% (166/208) alleles of MSP2, and 65.7% (65/99) for 3D7 family, but could not identify the type of any allele. While PCR-RFLP showed 25.3% higher genotyping efficiency than Nest-PCR. Moreover, this method could identify the allele types. CONCLUSION: PCR-RFLP genotyping technique is more efficient and specific than conventional Nest-PCR, and it is a convenient tool in the study on molecular epidemiology of malaria.

[Regulation mechanism of variation in Plasmodium falciparum var gene family].

This is a review on the new progress in the study of regulation mechanism of Plasmodium falciparum Var gene family. The mutually exclusive expression system caused expression only one in 60 var genes while others were silenced. It was regulated on the transcriptional level mainly through three pathways: non-coding DNA elements, chromatin structure and perinuclear localization.

[Expression and immunogenicity evaluation of ectodomain and subdomains of Plasmodium berghei apical membrane antigen 1].

OBJECTIVE: To express and evaluate the immunogenicity of ectodomain and its subdomains of Plasmodium berghei apical membrane antigen I (PbAMA-1). METHODS: Sequence of PbAMA-I gene was isolated from the genome of P. berghei, and was redesigned and divided into three overlapped fragments according to its subdomain structure. The codon-optimized DNA fragments of PbAMA-1 were synthesized and inserted into vector pET32a for expression in E. coli and the recombinant proteins were purified by Ni-NTA column, followed by refolding in vitro. Mice and rabbits were immunized with the recombinant proteins formulated with Freund adjuvant. Titer of the specific antibodies was detected by ELISA and IFA. The immunized mice were challenged by P. berghei to evaluate protective efficacy in vivo. RESULTS: The sequence of the PbAMA-1 gene was shown to be identical to that published before. PbAMA-1 sequence was redesigned via codon optimization and synthesized. Both ectodomain and its subdomains of PbAMA-1 were successfully expressed in E. coli after induction. The proteins were isolated with the purity of more than 90% after Ni column purification and refolding in vitro. Immunization of mice with the recombinant proteins induced high level of specific antibodies. The antibody titer to ectodomain E after the 3rd immunization showed a strong immunogenicity at (34.4+/-0.15) x 10(-4). The antibodies interacted with the parasites by indirect fluorescence. The immunized mice were partially protected from the challenge of P. berghei. CONCLUSION: The recombinant PbAMA-1 is highly immunogenic and induces protective immunity against the challenge of P. berghei.

[Preparation and characterization of monoclonal antibody specific to PfCP-2.9 chimeric protein of Plasmodium falciparum].

OBJECTIVE: To prepare and characterize monoclonal antibody against a malaria vaccine candidate, PfCP-2.9 chimeric protein of Plasmodium falciparum. METHODS: BALB/c mice were immunized with PfCP-2.9, and the spleen cells were used for fusion with SP2/0 cells. The monoclonal antibodies were analyzed by ELISA, Western blotting as well as growth inhibition assay. RESULT: A monoclonal antibody was obtained. It interacted with the PfCP-2.9 recombinant protein by ELISA and Western blotting. The interaction of the monoclonal antibody with the protein was reduction-sensitive, indicating that the antibody recognized a conformational epitope. Moreover, the antibody also recognized the cultured parasites of P. falciparum by indirect immunofluorescent antibody test (IFA). When tested by growth inhibition assay, the antibody significantly inhibited parasite growth in vitro of 56% inhibition rate at the antibody concentration of 0.3 mg/ml. CONCLUSION: A monoclonal antibody against PfCP-2.9 malaria vaccine candidate has been obtained, which recognizes a conformational epitope of the protein and natural protein.

[CTL response to pre-erythrocytic stage vaccine candidate of plasmodium falciparum in HLA-A*0201 transgenic mice detected by ELISPOT assay].

The importance of cytotoxic T-lymphocyte (CTL) against malaria parasite in pre-erythrocytic stage has been presented in relevant researches. In order to investigate whether one CTL epitope (YLNKIQNSL) involved in a chimeric pre-erythrocytic stage vaccine candidate of Plasmodium falciparum which was expressed and purified in the laboratory can stimulate in vivo CTL response, HLA-A*0201 transgenic mice were immunized with this vaccine candidate. Enzyme-linked immunosorbent spot (ELISPOT) assay was performed on the splenocytes from the immunized transgenic mice. Positive result indicated that this CTL epitope can be in vivo processed and correctly presented.

[Evaluation of immunogenicity and protection efficacy of the recombinant hypoxanthine-guanine-xanthine of plasmodium falciparum in mice].

OBJECTIVE: To investigate immunogenicity and protection efficacy of the recombinant hypoxanthine-guanine-xanthine (HGXPRT) of Plasmodium falciparum expressed in Pichia pastoris. METHODS: 35 BALB/c mice were divided randomly into five groups: HGXPRT+ISA720 experiment group, HGXPRT+Freund experiment group, ISA720 adjuvant control group, Freund adjuvant control group, and blank control group. BALB/c mice were subcutaneously immunized three times with the HGXPRT protein formulated by either Freund or ISA720 adjuvants at a three weeks interval. Mice were bled via tail vein at 2 weeks after each immunization. Specific antibodies were detected by ELISA as well as IFAT using cultured parasites. The immunized mice were challenged with 10(5) P. yoelii 10 days after the third immunization and parasitemia was monitored daily by examining Giemsa-stained thin film. RESULTS: Strong immune response was induced by the HGXPRT antigen formulated with the adjuvant. Antibody titers of more than 1:10(5) were detected after the third immunization while no specific antibody was detected in the mice immunized with adjuvants only. The antibodies against HGXPRT recognized the cultured parasite by IFAT. Four days after mice were challenged with P. yoelii, high parasitemia appeared in the two control groups, which were 24 h earlier than experiment groups. The mean parasitemia of HGXPRT+ISA720 experiment group (29.3%) was significantly lower than that of control groups (70.0%) (P<0.05). The mean parasitemia of HGXPRT+Freund experiment group (51.0%) was significantly lower than that of adjuvant control (60.7%) and blank control(70.0%) (P<0.05). CONCLUSION: HGXPRT of P. falciparum expressed in Pichia pastoris was highly immunogenic in mice. Antibody against the recombinant protein recognizes the cultured parasites, and immunization of mice with the recombinant protein provides partial protection against the challenge of P. yoelii.

[In vitro detection of immune sera to Plasmodium falciparum chimeric protein-2.9 for inhibition of parasite growth by LDH method].

OBJECTIVE: To investigate the in vitro inhibition effect of the rabbit serum immunized with P. falciparum chimeric protein on the parasites by lactate dehydrogenase (LDH) method. METHODS: Sera of rabbits immunized with P. falciparum chimeric protein were used as test sample, with an initial parasitemia of (0.4 +/- 0.1)% and 2% hematocrit suspension. After 40-42 h of cultivation, LDH assay and a traditional microscopical method were applied to obtain the parasite inhibition rate and infection rate of erythrocytes. RESULTS: Sera of rabbits immunized with the protein showed a strong in vitro inhibition effect on P. falciparum. There was no significant difference of inhibition rates detected by the two methods (97% and 100% respectively) (P>0.05). CONCLUSION: The LDH method applied in the in vitro inhibition assay for P. falciparum seems simple, reliable and has generated a satisfactory result.

[Preparation and characterization of monoclonal antibodies against erythrocyte-binding antigen 175 of Plasmodium falciparum].

OBJECTIVE: To prepare and characterize the monoclonal antibodies (mAbs) against erythrocyte-binding antigen 175 of Plasmodium falciparum (EBA-175). METHODS: BALB/ c mice were immunized with purified recombinant EBA-175 and mAbs against EBA-175 were prepared by means of hybridoma technique. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were employed for characterization of the mAbs. RESULTS: Six McAbs against EBA-175 antigen were obtained, 5 of which were identified as IgG1 and one as IgG2a. The titer of these aAbs was 1:12,800 to 1:25,600 in the ascites and 1:256 to 1:512 in supernatant, and ELISA demonstrated specific binding of the 4 mAbs (1F3, 2H5, 4A1 and 4H9)with Plasmodium falciparum. Three of these mAbs recognized the protein of EBA-175 as shown by Western blotting. CONCLUSION: Six hybridoma cell lines secreting the mAbs against EBA175 with high specificity are successfully established.

[Determination of free thiols in the chimeric protein PfCP-2.9 of Plasmodium falciparum].

OBJECTIVE: To determine the free thiols in the chimeric protein PfCP-2.9 of Plasmodium falciparum expressed by Pichia pastoris. METHODS: Two experiments of reverse phase HPLC and Ellman's reaction were applied to the PfCP-2.9 for the determination of its free thiols. For RP-HPLC analysis, three kinds of samples were tested: PfCP-2.9, dithiothreitol-reduced PfCP-2.9 and indoacetic acid-alkylated PfCP-2.9. RESULTS: Both experiments showed that there were no any free thiols present in the PfCP-2.9. CONCLUSION: The disulfide bonds between cysteine residues of PfCP-2.9 were formed completely.

[Influence of deleting 9 amino acid residues at N-terminus on immunogenicity of a Plasmodium falciparum chimeric protein].

Plasmodium falciparum chimeric protein 2 (PfCP-2), fused by erythrocytic stage antigens, AMA-1(III) and MSP1-19, is a potential vaccine candidate against malaria. However, the two-band pattern of this protein product in SDS-PAGE has some negative influence for the application of it for clinical tests. N-terminal sequence analysis of the product showed that the doublet had different N-terminus, with 9 amino acid deletion in the band with low molecular weight. Therefore, the gene was modified to generate a new construct, named PfCP-2.9, which was lack of these 9 residues at its N-terminus. Expression of PfCP-2.9 produced only one band. Moreover, the new construct was as same as the original product in the level of expression, conformation dependence on the disulfide bond, immunogenicity and inhibitory effect on the parasite growth in vitro.