Huan-Qin Zheng

Schistosomiasis is considered second only to malaria as the most devastating parasitic disease in tropical countries. Schistosome cercariae invade the host by penetrating the skin and migrate though the lungs and portal circulation to their final destination in the hepatic portal system and eventually the mesenteric veins. Previous studies have shown that the cytotoxic pathways that target schistosomulum in the lung-stage involve nitric oxide (NO) produced by macrophages. By contrast, skin-stage schistosomulas can evade clearance, indicating that they might be freed from macrophage NO-mediated cytotoxicity to achieve immune evasion; however, the critical molecules and mechanisms involved remain unknown. Recombinant SjCa8 (rSjCa8), an 8-kDa calcium-binding protein that is stage-specifically expressed in cercaria and early skin-stage schistosomulas of Schistosoma japonicum, was incubated with mouse RAW264.7 macrophages. Effects on macrophage proliferation were determined using Cell Counting Kit-8. Next, transwell assay was carried out to further investigate the role of rSjCa8 in macrophage migration. The effects of rSjCa8 on macrophage apoptosis were evaluated using confocal microscopy and flow cytometry. Additional impacts of rSjCa8 on NO release by lipopolysaccharide (LPS)-stimulated macrophages as well as the underlying mechanisms were explored using fluorescent probe, nitric oxide signaling pathway microarray, quantitative real-time PCR, mutagenesis, and neutralizing antibody approaches. rSjCa8 exhibited a striking inhibitory effect on macrophage migration, but did not markedly increase cell proliferation or apoptosis. Additionally, rSjCa8 potently inhibited NO release by LPS-stimulated macrophages in a dose- and time-dependent manner, and the inhibitory mechanism was closely associated with intracellular Ca2+ levels, the up-regulation of catalase expression, and the down-regulation of the expression of 47 genes, including Myc, Gadd45a, Txnip, Fas, Sod2, Nos2, and Hmgb1. Vaccination with rSjCa8 increased NO concentration in the challenging skin area of infected mice and reduced the number of migrated schistosomula after skin penetration by cercariae. Our findings indicate that SjCa8 might be a novel molecule that plays a critical role in immune evasion by S. japonicum cercaria during the process of skin penetration. The inhibitory impacts of rSjCa8 on macrophage migration and [Ca2+]i-dependent NO release suggest it might represent a novel vaccine candidate and chemotherapeutic target for the prevention and treatment of schistosomiasis.

Schistosomiasis japonicum remains a considerable economic and public health concern in China, the Philippines and Indonesia. Currently available measures to control the unique intermediate host Oncomelania hupensis are frequently associated with severe side effects. Previous studies have demonstrated that linalool-rich extracts from various plants exhibited promising biological activities including cytotoxic, anti-microbial and anti-parasitic properties. We identified the components of leaf extracts from Cinnamomum camphora by gas chromatography coupled to mass spectrometry (GC-MS) and investigated molluscicidal and larvicidal effects of linalool against O. hupensis and Schistosoma japonicium. The ultrastructural alterations in gills, salivary gland, stomach and hepatopancreas of snails were observed under the light microscope and transmission electron microscope, and lesions to tegument of cercaria were examined under a light microscope and fluorescence microscope. We then evaluated the effects of linalool on skin penetration and migration of schistosomula and adult survival by measurement of worm burden and egg counts in Balb/C mice infected with linalool-treated cercariae. In the present work, 44 components were identified from the leaf extracts of C. camphora, of which linalool was the most abundant constituent. Linalool exhibited the striking molluscicidal and larvicidal effects with LC50 = 0.25 mg/L for O. hupensis and LC50 = 0.07 mg/L for cercaria of S. japonicium. After exposure to linalool, damage to the gills and hepatopancreas of the snails, and to the tegument and body-tail joint of cercariae was apparent. In addition, linalool markedly reduced the recovered schistosomulum from mouse skin after challenge infection, and therefore decreased the worm burden in infected animals, but not fecundity of female adults of the parasite. Our findings indicated that linalool might be a novel chemotherapeutic agent against S. japonicium and the snail intermediate host.

To study the skin inflammation responses induced by heat shock protein 70 (rSj648/hsp70) from Schistosoma japonicum in BALB/c mice. BALB/c mice were injected with 20 microg of LPS in abdominal skin or 100 microg of rSj648/hsp70, meanwhile, the PBS-treated group was set as blank. On days 1, 2, 4, 7 after the immunization, dynamic changes of inflammation were observed by staining with HE; and the IFN-gamma mRNA expression was detected by real-time PCR. Results On day 1, the inflammation of the skins derived from mice injected with LPS was obviously, and then fell off gradually after day 1. Compared with the LPS-treated group, the inflammation responses induced by Sj648/hsp70 were longer and more intensive until until day 7. Heat shock protein 70 (rSj648/hsp70) induces high protection against schistosome-infection contributing to predominant Th1 reaction and the correlation with high expression of IFN-gamma.

To establish and maintain the life cycle of Clonorchis sinensis in laboratory. Adult worms and eggs of Clonorchis sinensis were collected from naturally infected cats. Eggs were ingested by freshwater snails in aquarium. When the cercariae were released from infected snails, they invaded into freshwater fishes. From the 30th day on after the release of cercariae, the infection rate and metacercariae density in freshwater fishes were determined. After 95 days the infected snails began shedding cercariae in a temperature range of 24.3 -37.2 degrees C, and no cercariae were found under 20 degrees C. The infection rate in the snails Parafossarulus striatulus and Alocinma longicornis was 12.5% and 18.0%, respectively. Metacercariae were found in fish at 30 days after cercariae infection, and matured metacercariae were detected in 45 days. The number of metacercariae per gram of fish meat in Pseudorasbora para, Ctenopharyngodon idellus, Rhodeus sinensis, Hypophthalmichthys nobilis, Cirrhinus molitorella, Carassius auratus, Cyprinus carpio and Oreochromis niloticus was 1 792, 16, 8, 6, 5, 4, 4, and 2, respectively. Rats and cats were fed with metacercariae from fish to receive adult worms. Life cycle of Clonorchis sinensis has been established and maintained in the laboratory.

To study the immunomodulation effect of the recombination Sj16 from Schistosoma japonicum (reSj16) on inflammation response of host. reSj16 expressed in pGEX-4T-1 was purified by GST purification kit and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectra (MS). The immunomodulation effect of reSj16 was observed by means of dimethylbenzene inducing mouse ear edema model, carrageenan inducing rat voix pedis swell model and acetic acid inducing mouse experiment peritonitis model. The soluble protein of reSjl6 was obtained and identified by SDS-PAGE and MS. reSjl6 1.0, 5.0, 10.0 microg/kg evidently suppressed the mouse ear edema induced by dimethyl-benzene, significantly mitigated the rat voix pedis swelling induced by carrageenan, and remarkably suppressed the increase of the capillary permeability of abdominal cavity in experiment peritonitis mouse model. The results further prove that Sj16 may be a potential immunosuppressive molecule and may have a notable effect on immunomodulation.