Comparison of ancient and modern Clonorchis sinensis based on ITS1 and ITS2 sequences.

In 1975, an ancient corpse buried in 167 BC was found at Jiangling County, Hubei Province of China. The eggs of Clonorchis sinensis found in the gall bladder of the corpse were preserved well. In the present paper, we extracted the genomic DNA from the ancient eggs and modern eggs, respectively, and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) at ribosomal RNA genes were studied. The results show that ITS2 sequences from the ancient sample were identical with those from modern samples, but in ITS1 differences in 15 nucleotide positions were found between the ancient and modern samples. The results demonstrated that it is possible to extract and sequence DNA from ancient parasite eggs. The ITS1 sequence obtained differed from all modern ones available to date. This might indicate sequence divergence through time, or might reflect a sequence polymorphism that may eventually be found also in modern samples.

[Studies on mechanism of protective immunity against infection of Schistosoma japonicum induced by Sj26 gene transfected dendritic cell].

OBJECTIVE: To explore the mechanism of protective immunity against Schistosoma japonicum infection induced by Sj26 gene transfected dendritic cell (DC). METHODS: 48 BALB/c mice were divided randomly into 4 groups with 12 each. The mice were injected through auricle for three times with Sj26 gene transfected DC (Group A), pcDNA3 transfected DC (Group B), untreated DC (Group C) and RPMI-1640 (Group D) respectively, and challenged with 40+/-2 cercariae of S. japonicum per mouse 2 weeks after the last immunization. Sera from mice were examined for IgG antibody, IFN-gamma and IL-4 by ELISA. Western blot was used for detecting specific anti-Sj26 IgG antibody. The production of IFN-gamma and IL-4 in the supernatant of spleen cells stimulated with soluble egg antigen (SEA) and ConA was quantified by sandwich ABC-ELISA. The proliferation of spleen cells were measured with MTT method. RESULTS: IgG antibody increased significantly in the mice of group A at 2 weeks after the last immunization (absorbency A491=0.117), higher than that of group B (A491=0.061) and group C (A491=0.058) (P<0.05). The Mr 26000 antigen of S. japonicum was strongly recognized by sera from group A by Western blot. The level of IL-4 in mice of each group showed no significant difference before and after immunization. The level of IFN-gamma in group A (101.4+/-4.9 pg/ml) was significantly higher than that before immunization (15.0+/-1.9 pg/ml) and that of group B (40.1+/-3.1 pg/ml) and group C (35.6+/-1.2 pg/ml) (P<0.01). The level of IFN-gamma in spleen cells from group A in response to ConA and SEA (171.2 and 70.8 pg/ml, respectively) was higher than that of group D (91 and 49.7 pg/ml, respectively) (P<0.01). The level of IL-4 in spleen cells from group A in response to ConA and SEA (79.7 and 50.7 pg/ml, respectively) was lower than that of group D (125.2 and 70.5 pg/ml, respectively) (P<0.01). The stimulating index of spleen cells from group A was 4.1 and 2.82 in response to ConA and SEA respectively, higher than that of other groups (compared with group D, P<0.05). CONCLUSION: Sj26 gene transfected dendritic cell induces predominant Th1 type immune response which might play a role in protection against S. japonicum infection.

Comparison of the role of dendritic cells and macrophages in inducing protective immunity against Schistosoma japonicum.

OBJECTIVE: To compare a potential role of dendritic cells (DCs) and macrophages in inducing protective immunity against infection with Schistosoma japonicum. METHODS: DCs and macrophages were pulsed in vitro with soluble egg antigen (SEA) of S. japonicum. BALB/c mice were injected three times with DCs or macrophages, either antigen-pulsed or not, and challenged with 40 +/- 2 cercariae of S. japonicum per mouse. Worms were collected 42 days later by portal perfusion of the mice and egg number of liver was calculated. To evaluate whether protective immunity had been induced by preparations of DCs or macrophages, the worm burden and fertility (eggs per female per mouse liver) were compared between the groups of mice. The antibody level against SEA was detected by ELISA. RESULTS: With respect to mice injected with untreated cells, numbers of worms and eggs per female worms were significantly reduced in the groups of mice having received pulsed DCs (26. 3% and 37.9%, respectively), or pulsed macrophages (22.0% and 30.7%). Untreated DCs and macrophages induced no significant effects. The antibody level against SEA rose in sera of all groups of mice up to 42 days after the challenge, but most pronounced in those immunized with pulsed DCs, although this was not significantly different from other groups. CONCLUSION: The results suggest that the protective immunity against S. japonicum might be induced by DCs to a higher extent than by macrophages after in vitro pulsing with egg antigen.

[Studies on the association of human leukocyte antigen class II alleles with advanced hepatosplenic schistosomiasis japonica].

OBJECTIVE: To study the association of human leukocyte antigen class II (HLA-II) alleles with genetic susceptibility and resistance to advanced hepatosplenic schistosomiasis japonica. METHODS: The allelic types of HLA-DRB1, DPA1, DQA1 and DQB1 were detected by polymerase chain reaction with sequence-specific primers (PCR-SSP) technique in 46 patients with advanced hepatosplenic schistosomiasis, characterized with extensive liver fibrosis. Another 43 subjects with chronic schistosomiasis were used as control. The statistical significance of differences in allelic frequencies was determined by chi2 test. RESULTS: The frequencies of HLA-DRB1 x 04, DPA1 x 0103, DQA1 x 0601, DQB1 x 0201 in advanced patients were markedly higher than those in control group, while the frequencies of HLA-DQA1 x 0501 and DQB1 x 0601 in control group were higher than those in advanced patients. CONCLUSION: The study indicated that HLA-DRB1 x 04, DPA1 x 0103. DQA1 x 0601 and DQB1 x 0201 showing a positive, statistically significant (P<0.05) association with advanced hepatosplenic schistosomiasis japonica may be the susceptible genes, whereas HLA-DQA1 x 0501 and DQBH1 x 0601 may be more relevant to a resistance to the disease.

[Expression of inducible nitric oxide synthase in the livers of mice infected with Schistosoma japonicum].

OBJECTIVE: To study the expression of inducible nitric oxide synthase (iNOS) in livers of mice infected with Schistosoma japonicum. METHODS: The livers of NMRI mice infected with S. japonicum were collected on day 21, 28, 38, 45 post infection (p.i.), total RNA of livers were extracted and kinetics of the mRNA expression of iNOS were detected by RT-PCR, the protein expression of iNOS was then confirmed by Western blotting and the distribution of iNOS in the infected liver was determined by immunohistochemical methods. RESULTS: The mRNA expression of iNOS was not detectable in the uninfected liver, iNOS mRNA expression was detected on day 21 p.i., the expression increased on day 28 p.i. and peaked on day 38 p.i., then decreased slightly on day 45 p.i. Western blotting showed an iNOS expression in the livers only on day 38, 45 p.i. IFA test showed that the expression of iNOS was mainly distributed in the granuloma of the livers. CONCLUSION: S. japonicum infection can induce the expression of iNOS in a time-dependent manner in the liver of the host, and eggs may be the main factor in inducing the expression.

[Distribution of inducible nitric oxide synthase in different stages of Schistosoma japonicum].

OBJECTIVE: To study the presence of iNOS in Schistosoma japonicum and demonstrate its distribution in different stages of this schistosome. METHODS: Cryostat sections for adult worms and paraffin sections for eggs in the liver of infected mouse, sporocysts and cercariae in snails were prepared, immunofluorescent test was performed to detect the presence of iNOS in adult worms, sporocysts, cercariae and miracidium, the distribution of this enzyme was observed in different stages of Schistosoma japonicum. Western blotting was used to further demonstrate the immunoreactivity to iNOS in adult worms. RESULTS: The results of immunofluorescent test showed that specific yellow-green fluorescence was mainly among subtugment of adult worms. Positive staining was also distributed on the surface of miracidium and its glands. For both sporocysts and cercariae, the majority of fluorescence was on their surface. Anti-iNOS antibody could recognize an apparent specific band in Western blotting of adult worm proteins, with a of Mr 210,000. CONCLUSION: There is an expression of iNOS-like enzyme in Schistosoma japonicum.