[The regulatory effect of dendritic cells on Th17 cell differentiation and function in mouse infected with Plasmodium yoelii].

Objective: To investigate the regulatory effect of dendritic cells (DCs) on Th17 cell differentiation and function during mouse infection with Plasmodium yoelii 17XL strain (Py17XL) and the underlying mechanisms. Methods: Twelve female BALB/c mice were randomly assigned into the infection group (Py17XL), the TLR4 blocking group (Py17XL + TLR4), TLR9 blocking group (Py17XL + TLR9), and TLR4 and TLR9 combined blocking group (Py17XL + TLR4+TLR9)(n=3 in each group). Mice in the Py17XL + TLR4 or the Py17XL + TLR9 group received intraperitoneal injection of 10 µg anti-TLR4 or 50 µg anti-TLR9 antibody (both 0.4 ml) to block DCs function at one day before infection. The Py17XL group received same volume of PBS. All groups were then given intraperitoneal injection of 1×10(6) red blood cells (RBCs) infected with Py17XL. The RBC infection rate was calculated on days 0, 3 and 5 after infection, and spleen cell suspension was prepared, in which the CD11c+TLR9+ and Th17 cells were counted by flow cytometry. The levels of IFN-γ and IL-10 in supernatant of spleen cell culture were determined by ELISA. Results: Flow cytometry showed that DCs were successfully blocked. On day 5 after infection, 28%,29%, 31% and 16.3% mice showed parasitemia in the Py17XL group, the Py17XL + TLR4 group, the Py17XL + TLR9 group, and the Py17XL + TLR4 + TLR9 group, respectively, and on day 7, the proportions were 43.3%, 47.5%, 32.5% and 8%. Mice in the Py17XL group and the Py17XL + TLR4 group all died, while those in other groups began to die from day 6. There was a slow rise of parasitemia rate in the Py17XL + TLR9 group and the Py17XL + TLR4 + TLR9 group compared with the Py17XL group, with a significant extension of survival to days 11 and 15. Results of flow cytometry showed that the proportions of Th17 cells were 1.2% and 1.44% in the Py17XL + TLR9 group and the Py17XL + TLR4 + TLR9 group on day 5, both sighificantly decreased compared with the Py17XL group (1.9%)(P < 0.05, P < 0.01). ELISA revealed that the levels of IFN-γ and IL-10 on day 5 in the Py17XL + TLR4 + TLR9 group [(232.4 ± 15.5) pg/ml and(1791.2 ± 58.2) pg/ml, respectively] were significantly higher than those in the Py17XL group[(90.7 ± 50.1) pg/ml and (962.6 ± 409.0) pg/ml](P < 0.05, P < 0.01). Conclusions: The differentiation and function of Th17 cells are regulated by DCs during Py17XL infection. Blockade of DCs decreases parasitemia and extends lifetime of mice. Further studies are needed to clarify the exact mechanisms.

[Exploration and Application of Discussion-based Teaching Method in Teaching of Medical Parasitology].

In this study, students majoring in Clinical Medicine were enrolled to explore the effect of discussion-based teaching method in the teaching of medical parasitology. One hundred and fifty-six students (with an entry year of 2011) in classes 1-3 received the discussion-based teaching while 153 students in classes 4-6 received traditional teaching. The effect of teaching was evaluated in terms of final examination score and questionnaire, and compared between the groups. The final examination score of students receiving the discussion-based teaching （86.1±6.6） was significantly higher than those receiving the traditional teaching（74.2±8.3）（P<0.05）. The discussion-based teaching method was graded as "excellent" by 89.1%（136/156）of the students, and was considered to be superior to the traditional teaching by 96.8%（151/156）of the students. The results indicate that the discussion-based teaching method can enhance interactions between participants, change the ways of thinking, and provide inspirations for learning and exploration.

[The ratio of Treg/Thl7 cells from mice infected with Plasmodium yoelii in the early stage of infection].

OBJECTIVE: To investigate the change of Treg/Th17 cell ratio in mice infected with Plasmodium yoelii 17XL in the early stage of infection. METHODS: Forty 6-8 week-old female BALB/c mice were randomly divided into infection group and Treg blockade group. All the mice were infected by intraperitoneal injection with 1 x 10(6) P. yoelii 17XL-parasitized erythrocytes. Mice in Treg blockade group were intraperitoneally injected with 1 mg CD25 mAb before infection and on day 1 post-infection, respectively. Three healthy mice served as control. Tail vein blood samples were collected in two groups each with 10 mice during day 1-7 post-infection. The parasitemia was monitored daily by examining Giemsa-stained thin film. The survival rate of infected mice was calculated. Before infection, on day 3 and 5 post-infection, the spleen was aseptically removed from each mouse in infection group (n=3) and Treg blockade group (n=3), and splenocyte suspensions were prepared. The proportion of Treg and Th17 cells were analyzed by flow cytometry. The ratio of Treg/Th17 cells was calculated and the relative analysis of the percentage of Treg and Th17 cells was conducted. RESULTS: Parasitemia appeared on day 2 post-infection in infection group. On day 5, the infection rate of P. yoelii in erythrocytes increased to (49.8±4.7)% with a survival rate of 60.0%. All mice died on day 6 post-infection. In Treg blockade group, P. yoelii 17XL-parasitized erythrocytes appeared on day 2 post-infection; the parasitemia was (10.0±3.5)% on day 5, and increased rapidly to 60.0% on day 7; all mice died on day 8. The percentages of Treg and Th17 cells reached (11.1±1.3)% and (16.7±3.3)% respectively on day 3 post-infection in infection group, and increased to (26.1±4.5)% and (19.6±2.6)% on day 5, considerably higher than that of the control (P<0.01). The ratio of Treg/Th17 cells was 0.7±0.1, 1.3±0.4 on day 3 and 5 post-infection in infection group, respectively, which was much lower than that of normal control (2.1±0.5) (P<0.01, P<0.05). On day 3 and 5 post-infection, the percentages of Treg cells in CD4+ T cells was (2.4±1.3)% and (4.3±2.9)%, respectively, and that of Th17 cells reached (26.1±2.2)% and (6.5±2.1)% in Treg blockade group. The ratio of Treg/Th17 cells in Treg blockade group was 0.1±0.1 and 0.7±0.4, respectively, which was considerably lower than that of the control (3.6±0.4)(P<0.01). The proportion of Treg cells in CD4+ T cells was positively correlated with that of Th17 cells (r=0.8166, P<0.01). CONCLUSION: The ratio of Treg/Th17 cells significantly decreases in the early stage of P. yoelii 17XL infection.

Genetic variation among Clonorchis sinensis isolates from different hosts and geographical locations revealed by sequence analysis of mitochondrial and ribosomal DNA regions.

The present study examined genetic variability among Clonorchis sinensis isolates from four different geographical localities (Guangzhou, Nanning, Jiamusi and Daqing) and host species (cats, dogs, human and rabbits) in Mainland China by sequence analyses of two mitochondrial DNA (mtDNA) genes, namely NADH dehydrogenase subunits 2, 5 (nad2 and nad5) and ribosomal internal transcribed spacer 1 (ITS1). A portion of the ITS1, nad2 (pnad2) and nad5 (pnad5) was amplified by polymerase chain reaction separately from adult C. sinensis individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of ITS1, pnad2 and pnad5 was 643, 666 and 771 bp, respectively. The intraspecific sequence variations within C. sinensis were 0-1.7% for ITS1, 0-1.4% for pnad2 and 0-0.9% for pnad5. The interspecific sequence variations within other zoonotic trematodes, which were published previously, were 4.5-84.9% for ITS1, 21.9-43.6% for pnad2 and 19.2-48.9% for pnad5. The A+T contents of the sequences were 45.26-45.88% (ITS1), 62.91-63.51% (pnad2) and 58.24-58.63% (pnad5). Phylogenetic analyses using ribosomal and mitochondrial sequence data set, with three different computational algorithms (Bayesian inference, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support. These findings demonstrated the existence of low-level intraspecific variations in ribosomal DNA (rDNA) and mtDNA sequences among C. sinensis isolates from four different regions and hosts in China and elucidated that mtDNA sequences and rDNA sequences provided reliable genetic markers for phylogenetic studies of zoonotic trematodes.

[Serum level of intercellular adhesion molecule-1 in patients with clonorchiasis and its impact on liver function].

OBJECTIVE: To investigate the serum level of intercellular adhesion molecule-1 (ICAM-1) in patients with clonorchiasis, and the relationship between ICAM-1 and liver function. METHODS: Fifty untreated clonorchiasis patients and 20 normal controls were subjected in the present study. Plasma levels of total bilirubin (TBIL), albumin (ALB) and alanine aminotransferase (ALT) were determined by automatic biochemical analyzer. The patients were divided into three experiment groups (I, II, and III) by Child-Pugh classification. Serum level of sICAM-1 was determined by double-antibody sandwich ELISA. Radioimmunoassay was used to detect the content of interleukin-4(IL-4), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in serum. LAL tripeptide substrate staining quantitative method was used to detect the level of bacterial lipopolysaccharide (LPS) in plasma. RESULTS: The level of sICAM-1, LPS, IL-4, IL-6, TNF-alpha, TBIL, and ALT [(729.34 +/- 75.67) microg/ml, (0.18 +/- 0.08) Eu/ml, (3.46 +/- 0.38) ng/ml, (223.48 +/- 46.90) pg/ml, (1.39 +/- 0.62) ng/ml, (15.45 +/- 10.81) micromol/L, and (39.25 +/- 8.82)IU/L, respectively] in serum or plasma of clonorchiasis patients were significantly higher than that of the control group [(269.15 +/- 38.21) microg/ml, (0.07 +/- 0.03) Eu/ml, (0.74 +/- 0.22) ng/ml, (106.06 +/- 32.96) pg/ml, (0.56 +/- 0.14) ng/ml, (6.31 +/- 4.70) micromol/L, (18.43 +/- 9.81) IU/L](P < 0.05 or P < 0.01). Plasma level of ALB [(28.35 +/- 5.38) g/L] was significantly lower than that of the control [(39.43 +/- 7.91) g/L] (P < 0.05). Correlation test showed that the sICAM-1 level in patients' sera was positively correlated with TBIL, ALT, and LPS (r = 0.662, 0.514, 0.499, P < 0.01), while negatively correlated with ALB (r = -0.423, P < 0.01). IL-4 level did not correlate with liver function parameters (P > 0.05). According to the Child-Pugh classification, the more serious the liver function damaged, the higher level of sICAM-1, LPS, IL-6 and TNF-alpha in the experiment groups. Significant differences were found between groups III and I (P < 0.01). CONCLUSION: Higher serum levels of sICAM-1, LPS, IL-6, and TNF-alpha in patients with clonorchiasis take part in the process of liver injury induced by Clonorchis sinensis.

[Study on molecular phylogeny of Schistosoma bovis based on mitochondrial DNA sequence and gene order].

OBJECTIVE: To determine the nucleotide sequence of the partial mitochondrial (mt) genome and the order of the mitochondrial protein-coding genes for Schistosoma bovis for analysis of possible phylogenetic position of this species in the genus Schistosoma. METHODS: The genomic DNA of adult worms were extracted by the GNT-K method. The target regions were amplified by PCR using a degenerated primer and specific primer. The PCR products were purified before ligating into the pGEM1 T-vector system. Recombinant plasmids were amplified in Escherichia coli, extracted and purified using routine methods. The nucleotide sequences were determined with an ABI PRISM 3100-Avant DNA sequencer using a BigDye Terminators v3.1 Cycle Sequencing Kit (Applied Bio-systems, CA, U.S.A.) with two T-vector specific primers (T7 and SP6). Positive colonies were sequenced with two internal specific primers to obtain the full sequence of each fragment on both strands by means of primer walking. Sequences of related schistosomes were retrieved from GenBank and aligned with our data. Gene trees were constructed using neighbor joining methods. RESULTS: The nucleotide sequence was determined and the gene order of this region in S. bovis was found as follows: NADHdehydrogenase4 (nad4)-trnQ (Gln)-trnK(Lys)-NADH dehydrogenase 3(nad3)-trnD (Asp)-NADH dehydrogenase 1(nad1). The gene order covering such region of S. bovis was similar to that of the African Schistosoma species, but strikingly different from the Asian species. Phylogenetic trees inferred from the alignment including partial nad4, nad3, partial nad1 and partial nad4+nad3+nad1 sequence for other 8 Schistosoma spp., respectively, revealed that S. bovis is placed proximally to S. haematobium in the African sub-group, which is identical with those placed by gene order in the African clade. CONCLUSION: The mtDNA analysis based on mitochondrial DNA sequence and the gene order strongly support the hypothesis that S. bovis belongs to the African schistosome clade rather than the Asian Schistosoma species.

Molecular genetic profiles among individual Clonorchis sinensis adults collected from cats in two geographic regions of China revealed by RAPD and MGE-PCR methods.

Clonorchis sinensis causes the important food-borne zoonosis, clonorchiasis, which is endemic in East Asia, especially in China mainly in Guangdong, Guangxi and Heilongjiang provinces and Korea. Although comparisons on isoenzymes and some molecular profiles of C. sinensis collected from different parts of China and Korea have been studied, few works on the genetic variation among the individuals from different regions of China has been reported. In the present study, individual adults of C. sinensis were collected from cats in two geographic locations (Guangdong province in the South and Heilongjiang province in the North) of China and 44 of them were examined by using random amplified polymorphic DNA (RAPD)-PCR and mobile genetic elements (MGEs)-PCR techniques to assess the individual genetic variability within and between the two groups of this parasite. Six arbitrary primers and two pairs of MGE primers were employed in the genomic DNA amplification. The molecular patterns showed significant polymorphism among the individuals. The RAPD data displayed that the similarity coefficient (SC) of the individuals within Heilongjiang group was much higher than that of the Guangdong group, which was further confirmed by MGE-PCR results. Individuals from Heilongjiang were found genetically closer with lesser polymorphisms than those collected from Guangdong province. These results demonstrated that RAPD and MGE-PCR techniques, particularly RAPD method, could be useful for investigating genetic variations among C. sinensis individuals. They may also indicate that the genetic variation of C. sinensis occurs in the subtropical region--Guangdong--faster than that in the cold-region--Heilongjiang province--due to more generations (life cycle) occurred.

[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.

[Studies on the relationship between the level of cytokine and liver function in patients with clonorchiasis sinensis].

OBJECTIVE: To explore the relationship between cytokine level and liver function among patients infected with Clonorchis sinensis. METHODS: 47 patients were divided into three groups according to the degree of Child-Pugh liver function grade: 20 in group A (3-4 scores), 15 in group B (5-6 scores) and 12 in group C (7-9 scores). Interleukin 2 (IL-2), soluble IL-2 receptor (sIL-2R), interleukin 8 (IL-8) and tumor necrosis factor (TNF-alpha) were examined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Automatic biochemical analyzer was employed for the determination of serum level of total bilirubin (TBL), albumin (ALB) and alanine aminotransferase (ALT). Data were analyzed with SAS statistic software. RESULTS: Serum levels of sIL-2R, IL-8 and TNF-alpha from patients were significantly higher than those obtained from healthy people (P<0.05, P<0.05, P<0.01), whereas the IL-2 level was significantly lower than the former (P<0.01). With the affected degree of the liver, serum levels of sIL-2R, IL-8 and TNF-alpha increased, in contrast to the decrease of IL-2 level. The differences were significant between groups A and C (P<0.05). The level of sIL-2R and TNF-alpha directly correlated with that of TBL (r=0.331 P<0.05, r=0.518 P<0.01) and ALT (r=0.475 P<0.01, r=0.285 P<0.05) respectively, but inversely correlated with the level of ALB (r=-0.319 P<0.05, r=-0.665 P<0.01). CONCLUSION: The infection of Clonorchis sinensis results in the reduction of cellular immune function of the patients. Certain relationship exists between serum cytokine level and liver function. Two cytokines, sIL-2R and TNF-alpha, are involved in the process of pathology.