Min Hou

Application of DNA-based diagnostics in detection of schistosomal DNA in early infection and after drug treatment

BackgroundResearch is now focused on identification of sensitive and specific diagnostic tests for early identification of schistosomal infection and evaluation of chemotherapy in field situations in China.ResultsThis study compared loop-mediated isothermal amplification (LAMP) with conventional PCR as DNA-based diagnostic techniques for the early detection of schistosomal DNA and the evaluation of chemotherapy. The results showed that both PCR and LAMP assays targeting a 301 base pair (bp) sequence of the highly repetitive retrotransposon, SjR2, amplified DNA from schistosomes but were unable to distinguish between schistosome species. LAMP and conventional PCR were shown to amplify the target sequence of the SjR2-pCR2.1 recombinant plasmid template with limits of detection of 10-4 ng and 10-2 ng, respectively, thus demonstrating the superior sensitivity of the LAMP method. Schistosoma japonicum DNA was detected in all serum samples obtained from the three experimental groups at 1 week post-infection by LAMP assay, while the rate of detection by conventional PCR ranged from 50% to 66%. The potential application of PCR and LAMP assays for the evaluation of artesunate and praziquantel chemotherapy was investigated. PCR was shown to be less sensitive for detection of schistosomal DNA in drug-treated rabbit sera than the LAMP method.ConclusionsThe data presented here indicate that LAMP is suitable for the detection of early infection in the groups primarily infected with Schistosoma japonicum, such as migrants, travellers, military personnel and the younger age groups. However, it is less suitable for evaluation of the efficacy of chemotherapy in the early stages because of its high sensitivity.

Deficiency in TLR2 but not in TLR4 impairs dendritic cells derived IL-10 responses to schistosome antigens.

The purpose of this study was to observe the diverse functions of Toll-like receptors (TLRs) in responses to specific schistosome antigens. Bone marrow-derived dendritic cells (BMDCs) from TLR2-deficient (TLR2(-/-)) or TLR4-deficient (TLR4(-/-)) mice were activated with soluble schistosomule antigen (SSA) or soluble egg antigen (SEA). TLR2 mRNA expression was significantly increased in B6 BMDCs following SEA stimulation. TLR2-deficient BMDCs showed enhanced MHCII expression following SSA and SEA stimulation. TLR2-deficient but not TLR4-deficient BMDC failed to produce IL-12p70 and IL-10 in response to schistosome antigens. TLR2-deficient BMDCs induced a stronger CD4(+) T cell proliferative response. IL-4 and IL-10 expression was inhibited in CD4(+) T cells primed with TLR2-deficient BMDCs, while enhanced in TLR4-deficient BMDCs-primed CD4(+) T cells. These results suggest that TLR2 is essential for the establishment of the DC production of IL-12p70 and IL-10.

TLR2 directing PD-L2 expression inhibit T cells response in Schistosoma japonicum infection.

Toll-like receptor 2 (TLR2) was shown to be an important immune receptor involved in the recognition of schistosome antigens, especially soluble egg antigen (SEA). In mice models with Schistosoma japonicum acute infection, we observed enhanced T cell-mediated immune responses in TLR2 knock out (TLR2−/−) mice compared with B6 mice. In Schistosoma japonicum chronic infection models, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) expression as well as TLR2 expression gradually increased in B6 mice, while only PD-L2 expression significantly decreased in TLR2−/− mice. Meanwhile, Programmed Death 1(PD-1) expression on CD4+T cells was down-regulated in TLR2−/− mice after a large number of egg appeared. We also found that stimulation with schistosome antigens, especially SEA, could up-regulate PD-L2 expression on BMDCs in a TLR2-dependent manner in vitro. Schistosome antigens primed-BMDCs with impaired expression of TLR2 or PD-L2 could induce CD4+T cells to produce low level of IL-10 or high level of IFN-γ. Our results indicated that TLR2 signaling can direct PD-L2 expression on DCs, which binds to PD-1 mainly on CD4+T cells, to help inhibit T cells response in Schistosoma japonicum infection.

Proteomic analysis of schistosomiasis japonica vaccine candidate antigens recognized by UV-attenuated cercariae-immunized porcine serum IgG2

Many studies have showed that the radiation-attenuated cercariae (RAC) vaccine could induce the high protection of laboratory animals to resist the schistosoma infection by cellular and humoral mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG2 antibody from RAC-vaccinated pigs or vaccination and challenge pigs. The antigens from the schistosomal soluble worm antigen preparation (SWAP) recognized by the porcine IgG2 antibody were obtained using immunoprecipitation technique. These antigens were separated by 2-D electrophoresis, and 116 spots were successfully identified by MALDI-TOF MS from about 400 putative spots in gels. Among these spots, 113 spots could match to the Schistosoma japonicum. These identified proteins in four groups were classified by Gene Ontology (Go) database, and the mainly functions of these proteins were involved in binding, catalytic activity (thioredoxin peroxidase-2, et al.), signal transduction class (MAP Kinase, et al.), cell process (the heat shock 70-kDa protein 9B, et al.), and the intracellular component (tektin, et al.). Our methods suggested that it was possible to pull-down the interesting proteins recognized by specific antibodies. Our results may provide new clues for exploring the mechanism of high protection induced by RAC and shed some light on the research for anti-schistosomiasis japonica vaccine.

PPAR‐γ agonist ameliorates liver pathology accompanied by increasing regulatory B and T cells in high‐fat‐diet mice

Peroxisome proliferator‐activated receptor (PPAR)‐γ plays critical roles in human metabolic disorders. However, the mechanism remains incompletely understood. Regulatory cells contribute to these metabolic improvements; therefore, whether PPAR‐γ agonist regulates regulatory cells was investigated.

SjTat-TPI facilitates adaptive T-cell responses and reduces hepatic pathology during Schistosoma japonicum infection in BALB/c mice

BackgroundSchistosomiasis is a kind of parasitic zoonoses which causes serious damage to public health and social development. China is one of the countries most affected by Schistosoma japonicum and an effective vaccine is still needed. In this study, we adopted Tat-mediated protein transduction technology to investigate the impact of different antigen presented approaches on host’s immune response and the potential protection against Schistosoma japonicum infection.ResultsWe successfully constructed the recombinant S. japonicum triosephosphate isomerase, Tat-TPI, as a vaccine candidate. Whether injected with Tat-TPI in foot pad or vaccinated with Tat-TPI in the back subcutaneously for three times, the draining popliteal lymph nodes and spleen both developed a stronger CD8+T response (Tc1) in mice. Not only that, but it also helped CD4+T cells to produce more IFN-γ than TPI immunisation. In addition, it could boost IgG production, especially IgG1 subclass. Most importantly, Tat-TPI immunisation led to the significant smaller area of a single egg granuloma in the livers as compared with TPI-vaccinated or control groups. However, the anti-infection efficiency induced by Tat-TPI was still restricted.ConclusionThis study indicated that immunisation with Tat-fused TPI could contribute to enhance CD4+T-cell response and decrease hepatic egg granulomatous area after S. japonicum infection though it did not achieve our expected protection against Schistosoma japonicum infection. The optimal vaccine strategy warrants further research.

Upregulated expression of cytotoxicity-related genes in IFN-γ knockout mice with Schistosoma japonicum infection.

It is well accepted that IFN-γ is important to the development of acquired resistance against murine schistosomiasis. However, the in vivo role of this immunoregulatory cytokine in helminth infection needs to be further investigated. In this study, parasite burden and host immune response were observed in IFN-γ knockout mice (IFNg KO) infected with Schistosoma japonicum for 6 weeks. The results suggested that deficiency in IFN-γ led to decreased egg burden in mice, with low schistosome-specific IgG antibody response and enhanced activation of T cells during acute infection. Microarray and qRT-PCR data analyses showed significant upregulation of some cytotoxicity-related genes, including those from the granzyme family, tumor necrosis factor, Fas Ligand, and chemokines, in the spleen cells of IFNg KO mice. Furthermore, CD8+ cells instead of NK cells of IFNg KO mice exhibited increased transcription of cytotoxic genes compared with WT mice. Additionally, Schistosoma japonicum-specific egg antigen immunization also could activate CD8+ T cells to upregulate the expression of cytotoxic genes in IFNg KO mice. Our data suggest that IFN-γ is not always a positive regulator of immune responses. In certain situations, the disruption of IFN-γ signaling may up-regulate the cytotoxic T-cell-mediated immune responses to the parasite.

PSMB8 regulates glioma cell migration, proliferation, and apoptosis through modulating ERK1/2 and PI3K/AKT signaling pathways

Glioma has been considered as one of the most aggressive and popular brain tumors of patients. It is essential to explore the mechanism of glioma. In this study, we established PSMB8 as a therapeutic target for glioma treatment. Expression of PSMB8 as well as Ki-67 was higher in glioma tissues demonstrated by western blot and immunohistochemistry. Then, the role of PSMB8 in migration and proliferation of glioma cells was investigated by conducting wound-healing, trans-well assay, cell counting kit (CCK)-8, flow cytometry assay and colony formation analysis. The data showed that interfering PSMB8 may inhibit the migration and proliferation of glioma cells by reducing expression of cyclin A, cyclin B1, cyclin D1, Vimentin, and N-cadherin, and by increasing expression of E-cadherin. Additionally, interfering PSMB8 may induce apoptosis of glioma cells by upregulating caspase-3 expression. Furthermore, these in vitro findings were validated in vivo and the ERK1/2 and PI3k/AKT signaling pathways were involved in PSMB8-triggered migration and proliferation of glioma cells. In an in vivo model, downregulation of PSMB8 suppressed tumor growth. In conclusion, PSMB8 is closely associated with migration, proliferation, and apoptosis of glioma cells, and might be considered as a novel prognostic indicator in patients with gliomas.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

AIM To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Protection and immunological study on two tetraspanin-derived vaccine candidates against schistosomiasis japonicum

Tetraspanins (TSPs) are proteins found on the surface of helminth parasites of the genus Schistosoma and are regarded as potentially protective antigens. The large extracellular loop of Schistosoma mansoni tetraspanin‐2, Sm‐TSP‐2, when fused to a thioredoxin partner and formulated with Freunds adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. It is well recognized that CD4+ T‐cell‐dependent immunity might play an important role against schistosomes; however, the contribution of CD8+ T cells against multicellular pathogen is still uncertain. The exogenous protein‐pulsed dendritic cells (DCs) can easily activate CD4+ T cells response, while CD8+ T cells response was relatively difficult to be induced. In this study, we evaluated the immunogenicity of TSP2HD antigen (hydrophilic domain of the S. japonicum tetraspanin‐2) and TAT (the protein transduction domain of HIV‐1)‐coupled TSP2HD protein. As TAT‐fused protein could promote major histocompatibility complex class I‐dependent antigen presentation in vitro, TAT‐TSP2HD‐pulsed DCs induced stronger proliferation of schistosome‐specific CD8+ T cells compared with DCs incubated with TSP2HD alone. Vaccination with TAT‐TSP2HD‐pulsed DCs in vivo could improve disease outcome in S. japonicum‐infected mice and was slightly superior to vaccination with DCs treated with TSP2HD. In summary, these data showed that TAT fusion proteins could help activate CD8+ cells and Th1 cells and provide part protection against schistosome.