T-B cell epitope peptides induce protective immunity against Mycoplasma pneumoniae respiratory tract infection in BALB/c mice.

Mycoplasma pneumoniae is the most common pathogen of community-acquired pneumonia in humans. Due to its high rates of antibiotic resistance, vaccination has become the best method to control the dissemination of M. pneumoniae. The recombinant carboxyl terminus of the P1 (P1C) protein is an immunodominant antigen, but it has negative effects such as poor stability and lower purity. In the current study, T-B epitopes of the P1C protein were predicted according to bioinformatics analysis and assessed for efficacy in peptide vaccination. BALB/c mice were subcutaneously inoculated with the T-B epitope peptides four times and then infected with M. pneumoniae through the respiratory tract. The results showed that the T-B epitope peptides of the P1C protein (P1C103-117, P1C155-169, P1C224-238 and P1C244-258) induced strong antigen-specific serum antibody responses and cellular immune responses with high levels of serum IgG, IgA antibodies and Th1-biased (IFN-γ and IL-2) cytokines. Immunization with T-B epitope peptides significantly reduced the M. pneumoniae burden and the degree of inflammation in the challenged mice. Furthermore, the levels of IFN-γ and TNF-α in the supernatants of lung homogenates were observably reduced compared to those in the PBS group. Overall, our findings demonstrate that T-B epitopes (P1C103-117, P1C155-169, P1C224-238 and P1C244-258) play significant roles in the P1C protein and can be used to induce powerful humoral and cellular immune responses to provide significant protection against M. pneumoniae pulmonary infection, which provides new insight into the design of potential multiepitope vaccines to prevent host infection by M. pneumoniae.

Protective effect of the ω-3 polyunsaturated fatty acids on the schistosomiasis liver fibrosis in mice.

This study aims to observe the effect of ω-3 polyunsaturated fatty acids on initiation and elimination of the schistosomiasis inflammatory response and liver fibrosis. The mice infected with the cercariae of Schistosoma japonicum (20 ± cercarie per mice) were separated randomly into several groups. After 60 days, liver tissue samples of all mice were sectioned. Hematoxylin-eosin (HE) staining, Masson staining, the enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FCM) were performed. Through HE and Masson staining, the size of egg (ovum) granuloma and the collagen deposited in mice's livers in ω-3 PUFAs and praziquantel mixed groups were less than that of model group and praziquantel treated group. The serum level of IL-13 and TNF-α were lower than that of model group and praziquantel treated group. The indicators of liver fibrosis, such as HA and LN in the group treated with ω-3 PUFAs and praziquantel before the release of soluble eggs antigen (SEA) into blood, were lower than that of model group and praziquantel treated group, respectively. The combined treatment of ω-3 polyunsaturated fatty acids and praziquantel conducted before the release of soluble eggs antigens into the blood decreases liver ovum granulomatous inflammation and fibrosis degree in the schistosomiasis. The mechanism of the ω-3 polyunsaturated fatty acid may be related to the adjustment of the anti-inflammatory and immune responses.

M13 phage peptide ZL4 exerts its targeted binding effect on schistosoma japonicum via alkaline phosphatase.

The present study was to determine the targeting effect of M13 phage peptide ZL4 (MppZL4) on Schistosoma japonicum (S.j). Mice infected with S.j were injected with MppZL4. Real-time PCR was used to detect the distribution and metabolism of MppZL4 in the livers and lungs of mice. In vivo refusion test was performed to detect the targeting of MppZL4. Western blotting was employed to determine the expression of MppZL4. Live imaging was used to detect the distribution of oligopeptide MppZL4. Immunohistochemistry was employed to determine MppZL4 location on adult S.j body surface. Gomori method was employed to detect the influence of oligopeptide MppZL4 on alkaline phosphatase activity. The distribution and metabolism of MppZL4 and M13KE are not significantly different from each other at each time point. The abundance of MppZL4 is changed as S.j migrates in mice. The targeted binding effect of MppZL4 varies at different stages. ZL4 oligopeptide targets S.j in mice. The specific binding sites of MppZL4 on S.j body are mainly located in syncytial cells. The binding sites of MppZL4 on S.j body surface might be ALP or ALP-related proteins. MppZL4 had targeted binding effect on S.j with its binding site being associated with proteins related to S.j alkaline phosphatase. S.j tegument had a specifically binding site with exogenous peptides, offering new means to explore the interactions between hosts and parasites. Additionally, MppZL4 can possibly be used as targeting molecules in worm-resistant drugs or as tracing molecules in imaging diagnosis technologies.

[Screening and analysis of peptides specifically binding to the schistosomulum tegument of Schistosoma japonicum].

OBJECTIVE: To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum, not cercaria, tegument of Schistosoma japonicum. METHODS: A 12 phage-display peptide library was screened with the S. japonicum schistosomula and cercariae as the target cells for biopanning by degrees, 15 positive clones were picked randomly and deduced by DNA sequencing. According the sequencing result, ELISA test, elution recovery test and immunohistochemical staining were performed to determine the specificity of the phages to the tegument. To further examine its binding properties, the positive peptide conjugated to RhB and recombinant pEGFP-C2 plasmid were similarly synthesized. RESULTS: After 3 rounds of biopanning, the phage recovery rate increased from 3.50 x 10(-5)% to 3.20 x 10(-2)%, indicating that the phage library was successfully enriched in the tegument of schistosomula. The analyzed sequences were identical with 3 peptide sequence of ZL6, ZL4 and ZL1. ELISA showed that the P/N value of MppZL4, MppZL6 and MppZL binding the schistosomulum membrane protein was 6.72, 3.65 and 2.22, while 1.58, 5.15 and 1.20 of binding the membrane protein of cercariae, respectively. Elution recovery test showed that the elution recovery rate of MppZL4 [(4.60 +/- 0.27) x 10(-2)%] was much higher than that of MppZL6 [(2.10 +/- 0.23) x 10(-3)%], MppZL1 [(1.20 +/- 0.28) x 10(-3)%] and M13KE [(1.30 +/- 0.60) x 10(-7)%] (P<0.01). Immunohistochemical staining showed that MppZL4 specifically bound to the tegument of schistosomula with a positive rate of 83.0% (83/100). Fluorescent microscopy revealed that the synthesized RhB-ZL4 bound to the tegument of schistosomula. The ZL4/pEGFP-C2 plasmid was introduced into juvenile S. japonicum and expressed in the parasite. CONCLUSION: The peptide of ZL4 specifically binds to the schistosomulum tegument but not to that of cercaria.

[Preparation of agar-paraffin double-embedded longitudinal sections of Schistosoma japonicum].

Schistosoma japonicum adults are pre-embedded in a double-layer agar and made the block, then dehydrated with alcohol, isobutyl alcohol and n-butyl alcohol. Various staining procedures can be conducted after conventional sectioning and dewaxing. Complete longitudinal serial sections of the pre-embedded worms can be obtained, and the desired sections can be easily located accurately.

[An efficient and accurate method for counting target molecules in phage-display peptide library].

The phage titer of samples representing the low, intermediate and high phage number was respectively determined by the double-layer agar plate (DLAP) method and real-time PCR assay. The two methods accurately measured the titer of samples. The plaques from about 1/3 double-agar layer plates could be used to determine the phage titer. The DLAP experiment should repeat 10 times with 10 microl sample each time, while the within-assay coefficient variation (CV) was 4.93%-30.38%. At the same time, the real-time PCR assay only repeated 3 times with 1 microl phage each time, while CV for within-assay ranged from 0.02% to 0.25%. Results indicated that real-time PCR is a simple and quick method for determining bacteriophage titer.

[Detection of Toxoplasma gondii DNA by loop-mediated isothermal amplification].

OBJECTIVE: To detect Toxoplasma gondii DNA by loop-mediated isothermal amplification (LAMP). METHODS: DNA was extracted by phenol-chloroform extraction from T. gondii tachyzoites. Four primers which recognized 6 distinct regions on the B1 gene of T. gondii were designed and used for LAMP assay. To evaluate the specificity of the method, Plasmodium vivax, P. falciparum, Pneumocystis carinii, Schistosoma japonicum, and mouse leucocytes were used gs controls. The parasite extract (T. gondii) was 10-fold serially diluted for evaluating the sensitivity of LAMP, and was amplified by LAMP. LAMP results were read with naked eye and analyzed by electrophoresis. RESULTS: After LAMP reaction, positive amplification was observed with T. gondii, but no positive signal was noted for the negative controls in the study. The sensitivity of LAMP assay reached up to 2-3 T. gondii tachyzoites/ml per reaction. CONCLUSION: LAMP assay shows proper specificity and sensitivity for the detection of T. gondii.