[Suppression effect of different stage antigens of Schistosoma japonicum on airway inflammation in a murine model of asthma].

OBJECTIVE: To investigate the effect of antigens of different stage Schistosoma japonicum on airway inflammation in a murine model of asthma. METHODS: 48 female BALB/c mice were randomly divided into eight groups. Mice in group A were given normal saline of equal volume as control. Group B was asthma model which was established by intraperitoneal and intranasal challenge with OVA. Mice in groups C, D and E were immunized with soluble egg antigen (SEA), soluble male worm antigen (SWA), and schistosomulum antigen (SSA) respectively 4 times in a week interval, followed by OVA sensitization as in group B 1 week after the final immunization. Mice in groups F, G, and H were immunized with SEA, SWA, and SSA respectively but sensitized and challenged with saline instead of OVA. 48 hours after asthma was induced, the mice were sacrificed. Leukocytes and eosinophils were counted in bronchoalveolar lavage fluid (BALF). The level of IL-5, IL-10 and IFN-gamma in BALF was detected. Pathologic changes in lung tissues were observed. RESULTS: Inflammation cells, especially eosinophils, appeared in airways of mice in groups B, C, D and E, but with much less number in groups C, D and E. No inflammation cells were seen in airways of group A mice. The number of leukocytes, eosinophils and level of IL-5 in BALF of group B [(98.4 +/- 16.1) x 10(4)/ml, (17.6 +/- 4.3) x 10(4)/ml, (197.9 +/- 36.5) pg/ml respectively] were significantly higher than those of group A [(8.2 +/- 1.1) x 10(4)/ml, (0.02 +/- 0.01) x 10(4)/ ml, (12.3 +/- 7.4) pg/ml], however the levels of IL-10 and IFN-gamma were significantly lower than that of group A (P < 0.05). The number of leukocytes, especially eosinophils, in BALF of groups C, D and E was significantly lower than that of group B. The level of IL-5 in BALF of groups C, D and E was significantly reduced, while that of IL-10 and IFN-gamma in BALF of the 3 groups was significantly higher than group B (P < 0.05). CONCLUSIONS: The immunization with S. japonicum antigens can effectively modulate the level of cytokines and inhibit the eosinophil infiltration and airway inflammation in asthmatic mice.

[Up-expression of PD-1-PD-L induced by immunization with SEA and SMWA of Schistosoma japonicum in mice].

OBJECTIVE: To explore the expression of PD-1-PD-L pathway of mice immunized with soluble egg antigen (SEA) or soluble male worm antigen (SMWA) of Schistosoma japonicum. METHODS: Eighteen BALB/c mice were randomly divided into three groups named as control group (A), SEA immunized group (B) and SMWA immunized group (C). Mice in groups B and C were subcutaneously immunized weekly with SEA (50 microg) and SMWA (50 microg) of S. japonicum respectively. After 4 times immunization, the expression of programmed death-1 (PD-1), programmed death-ligand1 (PD-L1) and PD-L2 in splenic cells was measured with flow cytometer. The expression of IL-4 and IFN-gamma in cultural suspension of splenic cells was detected by sandwich-ELISA after stimulation with ConA. RESULTS: The expression ratio of PD-1, PD-L1 and PD-L2 was extremely low in the control group, but increased after the immunization with SEA and SMWA. The expression ratio of PD-1 was (8.24 +/- 1.31)% in SEA immunized mice, higher than the mice immunized by SMWA [(6.08 +/- 1.28)%]. PD-L2 was much more elevated in SEA immunized mice [(5.26 +/- 1.73)%] while PD-L1 more significantly increased with SMWA immunization [(10.82 +/- 2.33)%]. In addition, the up-expression of PD-L1 was associated with the level of IFN-gamma and the expression of PD-L2 was associated with IL-4 secretion. CONCLUSION: The expression of PD-1-PDL was up-regulated in BALB/c mice immunized by SEA or SMWA of S. japonicum.

Improved Soluble Expression and Catalytic Activity of a Thermostable Esterase Using a High-Throughput Screening System Based on a Split-GFP Assembly.

The thermostable esterase Aaeo1 displays a low expression level and forms a great amount of inclusion bodies in E. coli. Herein, a split-GFP system was established in which the fluorescence intensity exhibited a good linear correlation with the soluble protein expression level and the esterase activity. In the primary high-throughput screening, the mutant library was screened by flow cytometry via detection of a split-GFP reporter. Then, through a secondary screening against esterase activity, two mutants with improved soluble expression and catalytic activity were obtained. The soluble expression of the mutant enzymes in E. coli was improved by 2-fold. The kcat/ Km values of the mutant enzymes were 2-fold higher than that of the parent. We explored the relationship between the amino acid mutations in the two mutants and the enzyme activity. The enzyme activity of mutant I51V-E170D was 4.5 times higher than that of the parent.

[Suppression of Schistosoma japonicum eggs on TNBS-induced colitis in mice].

OBJECTIVE: To study the suppression of Schistosoma japonicum eggs against the trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. METHODS: 50 female BALB/c mice (6-8 week-old) were randomly divided into normal control group, ethanol control group, schistosome egg immunized control group, TNBS-induced colitis group and TNBS-induced colitis with egg immunization group. In TNBS-induced colitis with egg immunization group, mice were immunized 4 times with 10,000 schistosome eggs by intraperitoneal injection with one-week interval. On day 6 after the last immunization the mice were induced by TNBS and the body weight, histological change on colon and level of cytokines of mice were observed in egg-immunized and -unimmunized colitis mice. RESULTS: The unimmunized mice developed significant inflammation in colon with bloody mucus feces and decreased body weight after TNBS induction. Distinct hyperemia, edema and transmural inflammatory infiltration accompanied with ulceration were shown in colon. The level of IFN-gamma was (3.47 +/- 0.87) ng/ml and IL-4 was (146.06 +/- 45.76) pg/ml. However, in egg-immunized mice, the inflammation was suppressed greatly and the body weight recovered soon after TNBS induction. The production of IL-4 was enhanced to (598.50 +/- 135.90) pg/ml, and IFN-gamma was significantly diminished to (1.53 +/- 0.51) ng/ml. CONCLUSION: S.japonicum eggs protect mice from colitis induced by TNBS.

[Production and identification of chicken egg yolk antibodies against soluble egg antigen of Schistosoma japonicum].

OBJECTIVE: To produce and purify egg yolk immunoglobulin against soluble egg antigen (SEA) of Schistosoma japonicum, and evaluate its specificity and sensitivity. METHODS: 25-week old hen was intravenously and subcutaneously immunized with SEA of Schistosoma japonicum for 4 times. Each hen was first immunized with 60 microg SEA and subsequent injections were performed at 10-day intervals with 30 microg SEA. IgY was extracted from eggs of hen 35 d after the first inoculation by WD (water-dilution) method, eggs from non-immunized hen were used as negative control. The protein concentration of IgY was measured by BCA method, and IgY was analyzed by SDS-PAGE and Western blotting. SEA-based ELISA was used to evaluate the specificity and sensitivity of the IgY. RESULTS: 61 mg IgY was extracted from one egg. The results of SDS-PAGE and Western blotting demonstrated that the IgY contained one major protein band with molecular weight of 130,000 and could be recognized by SEA. Specific IgY could be immediately detected by SDS-PAGE and ELISA in the eggs laid by the hens from 10 days after the first immunization. On day 31 after the primary immunization, the antibody titer reached 1:1 600. 2.4 ng/ml SEA was detected by IgY based-sandwich ELISA, which indicated a high sensitivity of the purified IgY. CONCLUSION: Anti-SEA IgY with high specificity and sensitivity has been obtained and purified.

[Protective efficacy of co-immunization with Sj26 DNA and recombinant protein vaccine against Schistosoma japonicum in mice].

OBJECTIVE: To study the protective efficacy of co-immunization with Schistosoma japonicum glutathione S-transferase (Sj26) DNA and recombinant Sj26 protein (rSj26 GST) vaccine against Schistosoma japonicum in BALB/c mice. METHODS: The Sj26 gene was cloned into eukaryotic expression vector pEGFP-N3 with enhanced green fluorescence protein. The recombinant plasmid pEGFP-Sj26 was transfected into baby hamster kidney (BHK) cells, fluorescent microscope and Western blotting were employed to identify the expressed products. Each mouse in co-immunization group was primed with plasmid pEGFP-Sj26, boosted 2 weeks later and immunized with rSj26 GST 4 weeks later. While each mouse in pEGFP-Sj26 group and rSj26 GST group was primed and boosted with pEGFP-Sj26 or rSj26 GST independently. Two weeks after last immunization, each mouse was challenged with 40 +/- 1 cercariae of S. japonicum Chinese strain. At the 45th day post-infection, mice were sacrificed and the worms were perfused from portal vein and the number of worms and eggs in liver tissue were counted. RESULTS: In BHK cells transfected with the recombinant plasmid pEGFP-Sj26, the expression of Sj26-EGFP fusion protein was confirmed by fluorescent microcopy and Western blotting. The worm reduction rate in co-immunized group was 50.8%, significantly higher than that in pEGFP-Sj26 group (28.0%, P < 0.01) and rSj26 GST group (25.5%, P < 0.01). Liver egg reduction rate in co-immunized group, pEGFP-Sj26 group and rSj26 GST group were 32.7%, 20.6% and 33.0% respectively. The number of eggs per female in liver of co-immunized mice and rSj26 GST group were significantly higher than that in control group (P < 0.01). CONCLUSION: Compared to the immunization with pEGFP-Sj26 or rSj26 GST alone, the co-immunization with pEGFP-Sj26 and rSj26 GST can enhance protective efficacy in BALB/c mice.

The effects of T cell deficiency on the development of worms and granuloma formation in mice infected with Schistosoma japonicum.

It is widely accepted that the immune response of the host attacks the parasite and the parasite appears to develop strategies to evade the assault. However, there is increasing evidence that the development of a parasite may be also positively influenced by the immune response of host. In this paper, we explore the effects of T cell deficiency on the development of the worms and granuloma formation in mice infected with cercariae of Schistosoma japonicum. T cell-deficient (nude) mice supported normal parasite survival and fecundity, but compared to normal mice delayed the worms' development (length and female fecundity) until 28 days after infection. However, these differences equaled out at 35 and 42 days. The nude mice apparently suppressed the size of granuloma in the livers around the eggs of S. japonicum. The granulomas were composed predominantly of neutrophils but with significantly fewer eosinophils in nude compared to normal mice. In addition, hepatocyte necrosis occurred in the vicinity of granulomas in nude but not normal mice. This is consistent with egg-granuloma formation in the host being dependent on T-lymphocyte functions and shows that the effect of T cell deficiency on the development of the worms is transitory in S. japonicum.

Schistosoma japonicum infection modulates the development of allergen-induced airway inflammation in mice.

Asthma, a chronic inflammatory disorder of the airways, is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. It has been shown that helminth infections including Schistosoma mansoni may modulate atopic diseases including asthma. In the present study, BALB/c mice were infected with bisexual and unisexual (male) S. japonicum, respectively, prior to ovalbumin (OVA) sensitization and challenge. Compared to mice with OVA sensitization/challenge alone, S. japonicum infection led to a significant decrease of eosinophil accumulation in bronchoalveolar lavage fluid (BALF) collected 48 h postchallenge, as well as to a marked reduction in inflammatory cell infiltration around the airways and pulmonary blood vessels. Compared to OVA-immunized uninfected mice, the level of OVA-specific serum IgE as well as interleukin (IL)-4 and IL-5 in BALF were reduced, but IL-10 was strongly elevated in mice with preexisting S. japonicum infection prior to OVA immunization. These results suggest that both bisexual and male S. japonicum infections may modulate the development of allergic asthma.

Schistosoma japonicum eggs modulate the activity of CD4+ CD25+ Tregs and prevent development of colitis in mice.

Crohn's disease (CD) is considered to be caused by a disorder of the immune system and helminth infections may interact with development of the disease. We induced colitis in mice by trinitrobenzenesulfonic acid (TNBS) and observed the effects of intraperitoneally injected eggs of Schistosoma japonicum on the course of the disease. The inflammation in the colon was reduced in egg-treated mice and secretion of IFN-gamma (a Th1 cytokine) by cultured spleen cells in vitro was greatly suppressed, and of IL-4, IL-5 and IL-10 (Th2 cytokines) significantly elevated after egg injection. Also, the percentage of regulatory T-cells (Tregs) was increased in the spleens of egg-exposed mice with TNBS-induced colitis compared to non-egg exposed animals. The data suggest that Tregs may be activated by S. japonicum eggs and play a role in restoring immune disorders in TNBS-induced colitis of mice.

Discovery of imidazolidine-2,4-dione-linked HIV protease inhibitors with activity against lopinavir-resistant mutant HIV.

A new series of HIV protease inhibitors has been designed and synthesized based on the combination of the (R)-(hydroxyethylamino)sulfonamide isostere and the cyclic urea component of lopinavir. The series was optimized by replacing the 6-membered cyclic urea linker with an imidazolidine-2,4-dione which readily underwent N-alkylation to incorporate various methylene-linked heterocycle groups that bind favorably in site 3 of HIV protease. Significant improvements compared to lopinavir were seen in cell culture activity versus wild-type virus (pNL4-3) and the lopinavir-resistant mutant virus A17 (generated by in vitro serial passage of HIV-1 (pNL4-3) in MT-4 cells). Select imidazolidine-2,4-dione containing PIs were also more effective at inhibiting highly resistant patient isolates Pt1 and Pt2 than lopinavir. Pharmacokinetic data collected for compounds in this series varied considerably when coadministered orally in the rat with an equal amount of ritonavir (5 mg/kg each). The AUC values ranged from 0.144 to 12.33 microg h/mL.