Efficacy of tribendimidine against Angiostrongylus cantonensis infection in the mice.

Angiostrongyliasis, also known as eosinophils meningitis, is caused by Angiostrongylus cantonensis parasites in the human central nervous system. Currently, the drug of choice for treatment of angiostrongyliasis is albendazole, but dead worm lysis causes severe inflammatory response, which leads to central nervous system damage. Tribendimidine, a broad-spectrum anti-helmintic drug developed in China, is a derivative of amidantel. This study was designed to test the efficacy of tribendimidine against A. cantonensis in mice. We treated 65 infected female BALB/c mice with tribendimidine or albendazole by oral route. We observed that tribendimidine at doses of 50, 100 and 200 mg/kg/day was effective, and the worm reduction rates were 54.8 %,77.4 %, and 100 % compared with the control group. In addition, the therapeutic effect of early tribendimidine treatment (7 days post-infection [PI]) was better than the late treatment (14 days PI), in comparison with the albendazole group (20 mg/kg/day). The index of therapeutic efficacy included body weight, neurological function, survival time, worm reduction, mRNA levels of proinflammatory cytokines in brain tissue, histopathological examination and electron microscopy scanning. The results showed that tribendimidine could kill the larvae of A. cantonensis in the mice model, and the worm's body wall was observed to be damaged. After treatment with tribendimidine, the survival conditions such as body weight and neurological function were improved, and brain inflammation was reduced in infected mice. This study showed a strong efficacy of tribendimidine against A. cantonensis and provided suitable alternative treatments to further explore its potential use in treatment of human angiostrongyliasis.

The mRNA level of the galectin-10 of Angiostrongylus cantonensis induced by reactive oxygen stress.

Galectin plays an important role in host-parasite interactions. In this study, we identified a novel gene encoding galectin-10 (AcGal-10) from the cDNA library of Angiostrongylus cantonensis and characterized its biological role in the parasite. Sequence and phylogeny analysis showed that AcGal-10 is related to other galectin family members with the conserved loci (H(84)-D(86)-R(88)-V(96)-N(98)-W(105)-E(108)-R(110)). The mRNA level of AcGal-10 was expressed in reactive oxygen stress radicals. We have identified two proteins of A. cantonensis galectin-10 gene, one of which was reported (AcGAL10-W) and the others is AcGAL-10-M. In addition, recombinant AcGal-10 (rAcGal-10) was constructed into the pGEX-4T-1 plasmid, purified, and finally confirmed by SDS-PAGE and LC-MS. Hemagglutination assay showed that the minimum concentration of rAcGAL10-W and rAcGAL10-M required for the hemagglutination of BALB/c mice erythrocyte was 25 µg/mL, and the carbohydrate-binding ability showed no difference between rAcGAL10-W and rAcGAL10-M. The mRNA levels of AcGal-10 were indeed expressed higher after stimulation with H(2)O(2) and recombinant A. cantonensis galectin-10. A mutation of AcGal-10 was also found, but there was no significant difference compared with the wild type. Furthermore, we also confirmed that recombinant AcGal-10 plays a role in the activation of the microglia. In conclusion, the report here showed that AcGal-10 may be an important molecule related to infection of A. cantonensis.

Molecular cloning and characterization of a matrix metalloproteinase, from Caenorhabditis elegans: employed to identify homologous protein from Angiostrongylus cantonensis.

Matrix metalloproteinases (MMPs) are a class of zinc-binding endopeptidases mainly responsible for degrading extracellular matrix constituent components, which also serve as effectors of leukocyte recruitment, cytotoxicity, cytokine and chemokine processing, and defensin activation involved in multiple mechanisms of immunomodulation. MMPs are a conserved proteolytic enzyme family participating in normal physiological function. In the present study, we have cloned a gene named CEMMP62 from Caenorhabditis elegans, the putative 62-kDa protein that contained 579 residues with MMP-conserved catalytic domain known as ZnMc_MMP and showed high identities with MMPs from other nematodes, and demonstrated that the recombinant CEMMP62 (rCEMMP62) expressed and purified from Escherichia coli could have weak proteolytic activity on swine gelatin; Western blot analysis revealed that sera from BALB/c mice immunized by recombinant protein could recognize excretory-secretary antigens from Angiostrongylus cantonensis third-stage larvae (L3). Also, the antiserum can recognize larval soluble antigens of L4 coming from mice (nonpermissive host) infected with A. cantonensis while it cannot recognize larval soluble antigens of L4 coming from rats (permissive host) infected with A. cantonensis. The results implied that probably CEMMP62 has homologous proteins which exist in A. cantonensis, and the potential MMP may play an important role in A. cantonensis infection and pathogenic process.

Efficacy of combined treatment with albendazole and baicalein against eosinophilic meningitis induced by Angiostrongylus cantonensis in mice.

Angiostrongylus cantonensis infection causes eosinophilic meningitis in humans. Baicalein is a flavonoid originally isolated from the roots of Scutellaria baicalensis Georgi. In this study we evaluated the efficacy of the combination of albendazole and baicalein for treating eosinophilic meningitis in BALB/c mice. Therapeutic efficacy included the survival time, body weight, neurological function, leucocyte and eosinophil counts, eotaxin concentration, matrix metalloproteinase-9 (MMP-9) activity, larval recovery and histopathological examination. The results showed that the combination of albendazole and baicalein was more effective than either drug administered singly. Combination therapy increased the survival time, decreased body weight loss, neurological dysfunction, leucocyte response, eotaxin concentration and MMP-9 activity. Our results suggest that the combination of albendazole and baicalein may exhibit synergistic beneficial effects in the treatment of eosinophilic meningitis induced by A. cantonensis.

[Cloning, expression and immunologic identification of C31B8.8 gene of Caenorhabditis elegans].

OBJECTIVE: To clone and express C31B8.8 gene of wild-type Caenorhabditis elegans, and study the immunological characteristic of the recombinant protein. METHODS: Total RNA was extracted from cultivated C. elegans and reversely transcribed into cDNA. C31B8.8 gene was amplified by PCR and cloned into pMD-18T vector for sequencing. The accurate sequence was subcloned into the expression vector pET-30a with (His) 6-tag. The recombinant plasmid was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was identified by using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and Western blotting 10 BALB/c mice were randomly divided into C31B.8-immunized group and PBS + adjuvant group Mice in C31B8.8-immunized group were immunized with 40 microg of purified C31B8.8 antigen formulated in Freund's adjuvant Mice in PBS + adjuvant group received only adjuvant emulsified with PBS. All the mice received four immunizations every week with the same dose of antigen. Serum samples were collected at pre-immunization and certain time after immunization and the antibody titer was analyzed by ELISA. The recombinant C31B8.8 protein and soluble components of Angiostrongylus cantonensis fourth-stage larvae were identified by Western blotting. RESULTS: The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. MALDI-TOF-MS and Western blotting analysis showed that the recombinant C31B8.8 protein was the target protein. Compared with PBS + adjuvant group, mice immunized with purified protein C31B8.8 produced higher level of IgG. The anti-C31B8.8 serum recognized recombinant C31B8.8 protein, and reacted with soluble antigens of A. cantonensis fourth-stage larvae. CONCLUSION: C elegans C31B8 gene shows certain immunogenicity and immunoreactivity, and the soluble antigens of A. cantonensis fourth-stage larvae can react with anti-C31B8.8 serum.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis.

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

Protection of guinea pigs against Leptospira interrogans serovar Lai by LipL21 DNA vaccine.

In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1(+). The guinea pigs were immunized with pcDNA3.1(+)-lipL21, pcDNA3.1(+) or PBS. Six weeks after the second immunization, the splenocytes were isolated to detect their proliferative ability by lymphocyte transformation experiments. In addition, microscopic agglutination test was used for quantitative detection of specific antibodies. The rest guinea pigs were challenged intraperitoneally with L. interogans sorevar Lai. Then, protective effect was evaluated on the basis of survival and histopathological lesions in the kidneys, lungs, and liver. The lipL21 gene was successfully expressed in COS-7 cells through recombinant pcDNA3.1(+)-lipL21. The titer of specific antibodies substantially increased, and the stimulation index of splenocytes increased significantly. Hence, the pcDNA3.1(+)-lipL21 could protect the immunized guinea pigs from homotypic Leptospira infection. Furthermore, no obvious pathologic changes were observed in the pcDNA3.1(+)-lipL21 immunized guinea pigs. The results showed that the protective effect with pathogenic strains of Leptospira was shared by LipL21 mediated through a plasmid vector. Consequently, these results indicated that the lipL21 DNA vaccine was a promising candidate for the prevention of leptospirosis.

Expression profile, localization of an 8-kDa calcium-binding protein from Schistosoma japonicum (SjCa8), and vaccine potential of recombinant SjCa8 (rSjCa8) against infections in mice.

Researches on genes expressed in a cercarial stage-specific manner may help us understand the molecular events and functions during schistosome invasion of skin. A genomic clone encoding 8-kDa calcium-binding protein (SjCa8) specifically expressed in cercariae and skin-stage schistosomulum (transformed within 3 h) was obtained from cercariae. Recombinant protein was expressed in vector pET32a (+) and purified using a Ni-NTA purification system. The target protein SjCa8 was determined by matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blot revealed SjCa8 can be detected in cercaria and skin-stage schistosomulum but not lung-stage schistosomulum, adult, or egg and was localized to head gland, penetration gland tubes, and penetration glands where Ca(2+) was abundant, and the cercarial tegument (but not tegument of tail) and body-tail junction. Furthermore, SjCa8 was interestingly detected in cercarial secretions. The characterization of SjCa8 indicated that it may undergo structural and physiological modifications, including repair of the surface membrane, changes in permeability that account for the loss of water tolerance, activities of calcium-depending enzymes, and immune signaling, etc. Furthermore, vaccination with rSjCa8 plus adjuvant induced protective effect with 50.39% worm reduction rate and significantly high hepatic and intestine egg reduction rates (54.16%, 50.63%, respectively), which is possibly mediated through an apparent induction of Th1-type immune response for strikingly high level of IgG2a and IgG2b developed in immunized C57BL/6 mice.