Omics-based investigation of pathological liver injury induced by Echinococcus multilocularis infection in mice.

BACKGROUND: Alveolar echinococcosis (AE) can cause severe liver injury and be fatal if left untreated. Currently, there are no effective therapeutic options for AE-induced liver injury. Therefore, by exploring the changes of gene proteins in mice with damaged liver, we attempted to identify the key molecules of liver damage, and provide data that will enable the development of drugs targeting hepatic AE. METHODS: BALB/c mice were inoculated with protoscoleces via the hepatic portal vein. Three months later, B-ultrasound examination and Hematoxylin-eosin (H&E) staining were used to confirm liver damage in mice. RNA sequencing and Liquid chromatography-mass spectrometry (LC-MS) were used to screen differentially expressed molecules associated with liver damage through bioinformatics, and Quantitative Real-Time PCR (qRT-PCR) was used to verify their expression. RESULTS: B-ultrasound examination showed liver lesions in the infected group, and H&E staining showed liver inflammation, fibrosis and liver necrosis. RNA sequencing and LC-MS results showed changes in the levels of more than 1000 genes and proteins, with upregulation of immune and inflammation pathways. By contrast, the downregulated genes and proteins were mostly involved in various metabolic reactions. Correlation analysis was conducted between the transcriptome data and proteome data. The results revealed 240 differentially expressed genes, of which 192 were upregulated, and 48 were downregulated. Many of these genes were involved in metabolic reactions, such as Catalase (Cat), fatty acid synthase (Fasn), and IL-16 genes, which may have relevance to liver injury. The results of qRT-PCR were consistent with those of bioinformatics analysis. CONCLUSIONS: The mechanisms of liver injury in mice infected with Echinococcus multilocularis are complex, involving abnormal metabolism, oxidative stress, inflammatory response, and many other factors. This study provides the data for preliminary exploration for the development of targeted therapies against AE.

Replantation of lamina spinous process ligament complex and miniature titanium plate shaping internal fixation in the treatment of tumors in the spinal canal.

OBJECTIVE: Purpose This study aims to explore the clinical efficacy of laminospinous process ligament complex reimplantation combined with mini-titanium plate fixation in the treatment of thoracolumbar intraspinal tumors. METHODS: A retrospective analysis was performed on 43 cases of intraspinal tumors treated with thoracolumbar intraspinal tumor resection from August 2018 to March 2021, and 27 cases underwent laminospinous process ligament complex reimplantation combined with micro titanium plate shaping. Fixation (laminar replantation group), and 16 patients underwent laminectomy combined with pedicle screw internal fixation (laminectomy group). The operation time, blood loss, drainage tube removal time, cerebrospinal fluid leakage, spinal instability, and the incidence of secondary spinal stenosis were compared between the two groups. The pain VAS score, ODI score, and modified Macnab at the last follow-up were compared between the two groups. And the laminar fusion rate of the laminoplasty group was measured. RESULTS: Both groups successfully completed the surgery and obtained complete follow-up. The incidence of cerebrospinal fluid leakage and secondary spinal canal stenosis in the laminectomy group was lower than that in the laminectomy group, and the difference was statistically significant (P < 0.05). There was no statistically significant difference in the incidence of spinal instability between the two groups (P > 0.05). The operation time and intraoperative blood loss in the laminectomy group were less than those in the laminectomy group, and the drainage tube removal time was earlier than that in the laminectomy group. The difference was statistically significant (P < 0.05). At the final follow-up, there was no statistically significant difference in the pain VAS score, ODI score, and modified Macnab between the two groups (P > 0.05), but they were all significantly improved compared with preoperative ones. Fusion evaluation was conducted on the laminoplasty group. Two years after surgery, the fusion rate was 97.56% (40/41). CONCLUSIONS: The application of laminospinous process ligament complex reimplantation combined with mini titanium plate fixation during thoracolumbar intraspinal tumor resection can effectively reconstruct the spinal canal and posterior column structure, reduce the incidence of cerebrospinal fluid leakage and secondary spinal stenosis. The laminar fusion rate is high.

Single-Cell RNA Sequencing Reveals Unique Alterations in the Immune Panorama and Treg Subpopulations in Mice during the Late Stages of Echinococcus granulosus Infection.

Cystic echinococcosis (CE) is a common zoonotic parasitic disease that seriously impacts public health. However, the full spectrum of immune cell changes in Echinococcus granulosus infection, especially the negative immune regulation of subpopulations of regulatory T (Treg) cells, are not yet well understood. In this study, we used single-cell RNA sequencing and immunome repertoire (IR) sequencing to analyze 53,298 cells from the spleens and peripheral blood mononuclear cells (PBMCs) of healthy and E. granulosus-infected mice. We used immunofluorescence combined with RNA fluorescence in situ hybridization and quantitative real-time PCR to verify the sequencing results. Our results showed tissue-specific immune system alterations in mice infected with E. granulosus. E. granulosus-infected mice induced a subpopulation of CD4+ cells with type I interferon production potential. Furthermore, there were six different Treg cell subpopulations in vivo at three stages of differentiation, and Treg subpopulations of different classes and different stages of differentiation showed tissue specificity. After infection, the Lag3hi Treg and Gpr83+Igfbp4+ naive Treg subpopulations were specifically induced in PBMCs and the spleen, respectively. Furthermore, T follicular helper 2 (Tfh2) cells with high expression of Cxxc5 and Spock2 were found in E. granulosus-infected mice. Our data uncovered changes in the full spectrum of immune cells in mice following the late stages of E. granulosus infection, including subpopulations of cells that have not been emphasized in previous studies. These results further enrich the study of the bidirectional immunomodulatory mechanism and offer a different perspective for subsequent studies of infection in E. granulosus.

Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep.

Echinococcosis is a common human and animal parasitic disease that seriously endangers human health and animal husbandry. Although studies have been conducted on vaccines for echinococcosis, to date, there is no human vaccine available for use. One of the main reasons for this is the lack of in-depth research on basic immunization with vaccines. Our previous results confirmed that recombinant antigen P29 (rEg.P29) induced more than 90% immune protection in both mice and sheep, but data on its induction of sheep-associated cellular immune responses are lacking. In this study, we investigated the changes in CD4+ T cells, CD8+ T cells, and antigen-specific cytokines IFN-γ, IL-4, and IL-17A after rEg.P29 immunization using enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and flow cytometry to investigate the cellular immune response induced by rEg.P29 in sheep. It was found that rEg.P29 immunization did not affect the percentage of CD4+ and CD8+ T cells in peripheral blood mononuclear cells (PBMCs), and was able to stimulate the proliferation of CD4+ and CD8+ T cells after immunization in vitro. Importantly, the results of both ELISPOT and ELISA showed that rEg.P29 can induce the production of the specific cytokines IFN-γ and IL-17A, and flow cytometry verified that rEg.P29 can induce the expression of IFN-γ in CD4+ and CD8+ T cells and IL-17A in CD4+ T cells; however, no IL-4 expression was observed. These results indicate that rEg.P29 can induce Th1, Th17, and Tc1 cellular immune responses in sheep against echinococcosis infection, providing theoretical support for the translation of rEg.P29 vaccine applications.

Identification of a dominant murine T-cell epitope in recombinant protein P29 from Echinococcus granulosus

Echinococcus granulosus causes echinococcosis, an important zoonotic disease worldwide and a major public health issue. Vaccination is an economical and practical approach for controlling E. granulosus. We have previously revealed that a recombinant protein P29 (rEg.P29) is a good vaccine candidate against E. granulosus. However, T cell immunogenic epitopes have not been identified. In the present study, we use rEg.P29-immunized mice as models to screen immunogenic epitopes for the construction of a novel multi-epitope vaccine. We search for immunodominant epitopes from an overlapping peptide library to screen the peptides of rEg.P29. Our results confirm that rEg.P29 immunization in mice elicits the activation of T cells and induces cellular immune responses. Further analyses show that a T cell epitope within amino acids 86­100 of rEg.P29 elicits significant antigen-specific IFN-γ production in CD4+ and CD8+ T cells and promotes specific T-cell activation and proliferation. Collectively, these results provide a reference for the construction of a novel vaccine against broad E. granulosus genotypes based on epitopes of rEg.P29.

Expression profiling of exosomal miRNAs derived from different stages of infection in mice infected with Echinococcus granulosus protoscoleces using high-throughput sequencing.

Echinococcosis is a worldwide zoonosis. The mechanism of the establishment, growth, and persistence of parasites in the host has not been fully understood. Exosomes are found to be a way of information exchange between parasites and hosts. They exist in various body fluids widely. There are few studies on host-derived exosomes and their miRNA expression profiles at different infection time points. In this study, BALB/c mice were intraperitoneally infected with protricercariae. Exosomes were extracted from plasma (0, 3, 9, and 20 weeks post infection), and the expression profiles of exosome miRNA in the peripheral blood of mice were determined using RNA-sequencing. Compared to the 0 week groups, 24, 35, and 22 differentially expressed miRNAs were detected in infected mouse at the three infection stages, respectively. The results showed that there were significant differences in the miRNAs of exosomes at different infection time points. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to annotate the different miRNAs. The results showed that the biological pathways of parasites changed significantly at different stages of infection, with many significant and abundant pathways involved in cell differentiation, inflammation, and immune response, such as MAPK signaling pathway, Th17 cell differentiation, Wnt signaling pathway, FoxO signaling pathway, Notch signaling pathway, etc. These results suggest that miRNA may be an important regulator of interactions between Echinococcus granulosus and host. The data provided here provide valuable information to increase understanding of the regulatory function of microRNAs in the host microenvironment and the mechanism of host-parasite interaction. This may help us to find targets for Echinococcus granulosus to escape host immune attack and control Echinococcus granulosus infection in the future.

Proteomic Analysis of Plasma-Derived Extracellular Vesicles From Mice With Echinococcus granulosus at Different Infection Stages and Their Immunomodulatory Functions.

The globally distributed cystic echinococcosis (CE) is caused by the larval stage of Echinococcus granulosus (E. granulosus), a cosmopolitan and zoonotic disease with potentially life-threatening complications in humans. The emerging roles for extracellular vesicles (EVs) in parasitic infection include transferring proteins and modifying host cell gene expression to modulate host immune responses. Few studies focused on the host-derived EVs and its protein profiles. We focused on the EVs from mouse infected with E. granulosus at different stages. ExoQuick kit was used for isolating EVs from mouse plasma and ExoEasy Maxi kit was used for isolating protoscolex culture supernatant (PCS) and hydatid cyst fluid (HCF). Firstly, EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and immunoblot. Secondly, the proteins of plasma EVs were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting LC-MS/MS data were processed using Maxquant search engine (v 1.5.2.8). Tandem mass spectra were researched against the mice and E. granulosus proteins database in the NCBI. The differentially expressed proteins are performed by proteomic label-free quantitative analysis and bioinformatics. Thirdly, in vitro experiment, the results of co-culture of plasma EVs and spleen mononuclear cells showed that 7W-EVs can increase the relative abundance of regulatory T (Treg) cells and IL-10. We further verified that EVs can be internalized by CD4+ and CD8+ T cells, B cells, and myeloid-derived suppressor cells (MDSC). These results implied host-derived EVs are multidirectional immune modulators. The findings can contribute to a better understanding of the role of host-derived EVs which are the optimal vehicle to transfer important cargo into host immune system. In addition, we have found several important proteins associated with E. granulosus and identified in infected mouse plasma at different stages. Furthermore, our study further highlighted the proteomics and immunological function of EVs from mouse infected with E. granulosus protoscoleces at different infection stages. We have laid a solid foundation for the role of EVs in cystic echinococcosis in the future research and supplemented a unique dataset for this E. granulosus.

Myeloid-derived suppressor cells exert immunosuppressive function on the T helper 2 in mice infected with Echinococcus granulosus.

Cystic echinococcosis (CE) is a worldwide hazardous zoonotic parasitosis caused by Echinococcus granulosus. CE development involves complex immunological mechanisms, including participation of multiple immune cells and effector molecules. Myeloid-derived suppressor cells (MDSCs) are known to be involved in chronic and acute inflammatory conditions. In this study, we aimed to characterize the immune function of MDSCs in CE to improve the understanding, prevention and treatment of CE. Our results indicated that MDSCs overexpressing Ly6C and Ly6G inhibit the formation and activity of T helper 2 cells in a NO-dependent manner during E. granulosus infection.

Proteomic Analysis on Exosomes Derived from Patients' Sera Infected with Echinococcus granulosus.

Cystic echinococcosis (CE), a zoonotic disease caused by Echinococcus granulosus at the larval stage, predominantly develops in the liver and lungs of intermediate hosts and eventually results in organ malfunction or even death. The interaction between E. granulosus and human body is incompletely understood. Exosomes are nanosized particles ubiquitously present in human body fluids. Exosomes carry biomolecules that facilitate communication between cells. To the best of our knowledge, the role of exosomes in patients with CE is not reported. Here, we isolated exosomes from the sera of patients with CE (CE-exo) and healthy donors and subjected them to liquid chromatography-tandem mass spectrometry analysis. Proteomic analysis identified 49 proteins specifically expressed in CE-exo, including 4 proteins of parasitic origin. The most valuable parasitic proteins included tubulin alpha-1C chain and histone H4. And 8 proteins were differentially regulated in CE-exo (fold change>1.5), as analyzed with bioinformatic methods such as annotation and functional enrichment analyses. These findings may improve our understanding about the interaction between E. granulosus and human body, and may contribute to the diagnosis and prevention of CE.

Extracellular vesicles derived from Echinococcus granulosus hydatid cyst fluid from patients: isolation, characterization and evaluation of immunomodulatory functions on T cells.

Cystic echinococcosis is a chronic and complex zoonotic disease. The mechanisms underlying the parasite's establishment, growth and persistence are not completely understood, and are thought be modulated by a crosstalk through extracellular vesicles. Here, EVs were isolated from the hydatid cyst fluid of patients with cystic echinococcosis and protoscolex culture supernatant. Proteomic analysis of these EVs revealed several parasite- and human-derived proteins. Very few studies have performed proteomic analysis of EVs isolated from HCF and PCS. Our proteomic analysis of the EVs derived from HCF and PCS facilitated identification of 1175 proteins, wherein 1026 and 38 proteins were exclusively identified in the EVs derived from HCF (HCF-EVs) and PCS (PCS-EVs), respectively, and 111 proteins were shared in both. The results of co-culture of PCS-EVs with murine peripheral blood mononuclear cells showed that PCS-EVs significantly regulated T lymphocyte functions in a dose-dependent manner. Collectively, our results provide valuable information on parasite survival strategies and new insights into the role of these EVs in the establishment and persistence of hydatid cysts.

Impairment of peripheral Vdelta2 T cells in human cystic echinococcosis.

Cystic echinococcosis (CE) induced by metacestodes (larval stages) of Echinococcus granulosus (E.granulosus) represents a severe endemic disease worldwide. Gamma delta (γδ) T cells, one of innate immune cells, play pivotal role in pathogenic infections. However, whether γδ T cells are involved in CE remains unclear. This study firstly investigated the role of peripheral γδ T cells in CE. The results showed that the percentage of peripheral γδ T cells from CE patients was decreased, compared with healthy controls (HC) (p < 0.01). This decrease was primarily due to a reduction in Vδ2 subset. Furthermore, Vδ2 T cells in CE expressed lower Natural Killer Group 2D (NKG2D) (p < 0.01). The abundance of Vδ2 T cells correlated negatively with cyst burden. To further identify the function of decreased Vδ2 T cells in CE, proliferation rate, cytokine secretion and cytotoxin were detected subsequently in vitro. As a result, the proliferation rate of Vδ2 cells in CE patients was lower than that in HC (p < 0.01). Meanwhile, Vδ2 T cells from CE patients released significantly decreased interferon (IFN)-γ, compared with HC (p < 0.05). Moreover, the levels of perforin and granzyme B of Vδ2 T cells from the patients were decreased significantly (p < 0.05), suggesting impaired cytotoxin generation of Vδ2 cells in CE. Collectively, our findings indicated that circulating Vδ2 T cells in CE was impaired, and these aberrations may contribute to disease pathogenesis.

Immunoprotection of recombinant Eg.myophilin against Echinococcus granulosus infection in sheep.

The aims of the present study were to investigate the immunoprotection of recombinant Echinococcus granulosus myophilin (rEg.myophilin) against the establishment of a challenge oral infection with E. granulosus eggs, as well as to determine the mechanisms underlying this protection. Sheep were subcutaneously immunized two times with rEg.myophilin, followed by the challenge with E. granulosus eggs orally. The animals were sacrificed 44 weeks after infection and the immunoglobulin (Ig) and cytokine levels were analyzed using ELISA. The results identified significant changes in several indexes of animal immune response subsequent to immunization with rEg.myophilin. These changes included reduced number of formed cysts, as well as elevated levels of IgG, IgA and cytokines. The present data suggest that immunization with rEg.myophilin in sheep can successfully reduce the formation of cysts caused by challenge E. granulosus infection and stimulate immune response, suggesting that rEg.myophilin a has potential value as a candidate vaccine against E. granulosus.

Immunoprotection of recombinant Eg.P29 against Echinococcus granulosus in sheep.

OBJECTIVE: This study aims to investigate the immunoprotection of recombinant Eg.P29 (rEg.P29) vaccine and analyze the underlying mechanism in sheep. METHODS: Three groups of male sheep were immunized subcutaneously with rEg.P29 and PBS, Freund's complete adjuvant as controls, respectively. After prime-boost vaccination, the sheep were challenged with encapsulated Echinococcus granulosus eggs. The percentage of protection in sheep was determined 36 weeks after the infection. Humoral immune response was analyzed for specific IgG, IgG1, IgG2, IgM and IgE levels. Moreover, cytokines including interferon (IFN)-γ, interleukin (IL)-2, IL-4,and IL-10 were also evaluated. RESULTS: Immunization with rEg.P29 induced protective immune responses up to 94.5 %, compared with immunoadjuvant group. The levels of specific IgG, IgG1, IgG2, and IgE as well as IFN-γ, IL-2, and IL-4 significantly increased after two immunizations (P < 0.05); however, the levels of IgM and IL-10 did not show difference. CONCLUSION: rEg.P29 showed Immunoprotection and induced Th1 and Th2 immune responses; hence, rEg.P29 is a potential vaccine for E. granulosus infection.

Mechanism of protective immunity by vaccination with recombinant Echinococcus granulosus glutathione S-transferase (Chinese strain) in mice.

The aim of this study was to investigate the immunoprotective effects of recombinant Echinococcus granulosus glutathione S-transferase (rEgGST) against the development of protoscolices (PSCs), and to determine the mechanisms underlying this protection. ICR mice were subcutaneously immunized three times with rEgGST at weeks 0, 2 and 4, followed by the intraperitoneal administration of E. granulosus PSCs at week 10. Six mice in each group were sacrificed at 0, 2, 4, 6, 10, 18 and 30 weeks following the initial vaccination in order to observe the macroscopic and microscopic effects of parasite development. Various analyses were subsequently conducted, including determination of the levels of immunoglobulins (Igs) and cytokines. Significant differences were observed a number of indices of immune response following immunization with rEgGST. These included reduced cyst formation and elevated levels of IgG1, IgG2a, IgG3, IL-2, IL-4, IL-10 and IFN-γ, which indicated an increased percentage of immune helper cells. The results of the present study suggest that immunization with rEgGST in mice is able to successfully reduce the PSC-induced formation of cysts and to stimulate an immune response, suggesting that rEgGST possesses potential value as a candidate vaccine for PSC infection.

Baicalin inhibits TLR7/MYD88 signaling pathway activation to suppress lung inflammation in mice infected with influenza A virus.

The present study aimed to investigate the protective effects and underlying mechanisms of baicalin on imprinting control region mice infected with influenza A/FM/1/47 (H1N1) virus. Oral administration of baicalin into mice infected with H1N1 prevented death, increased the mean time to death and inhibited lung index and lung consolidation. Subsequently, fluorescence quantitative polymerase chain reaction was used to assess the mRNA expression of toll-like receptor 7 (TLR7) and myeloid differentiation primary response gene 88 (MYD88), and western blot analysis was used to determine the expression of phosphorylated nuclear factor κB (NF-κB)-P65 and c-jun/activator protein 1 (AP-1). An enzyme-linked immunosorbent assay was applied to test for the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß and IL-6, in the lung tissue. The findings indicated that baicalin downregulated the mRNA expression of TLR7 and MYD88, significantly downregulated the protein expression of NF-κB-P65 and AP-1 and also inhibited the secretion of TNF-α, IL-1ß and IL-6. In conclusion, baicalin effectively reduced the pathological damage and inflammation of the lungs by downregulating the TLR7/MYD88-mediated signaling pathway.

[Preliminary study on the difference in immune protection between Echinococcus granulosus recombinant ferritin and recombinant mMDH].

OBJECTIVE: To evaluate the immunoprotective activity of the Egrecombinant ferritin and Egrecombinant mMDH proteins in mice. METHODS: Thirty ICR mice were divided into 3 groups and immunized by injection of adjuvant-emulsified rEgferritin, rEgmMDH and PBS, respectively, in multiple spots at back, for 3 times with an interval of 2 wk. Two weeks after the last immunization, the mice of the 3 groups were infected intraperitoneally with 0.1 ml suspension containing about 1 500 Echinococcus granulosus (Eg) protoscoleces. The mice were sacrificed 22 wk after infection and the Eg cysts were collected and measured. Spleens were taken for detecting CD4+ T cells and CD8+ T cells and ratio calculated. RESULTS: Eg cysts were found in 30% (3/10) of the mice in the rEgferritin group with 5 cysts altogether; cysts were received in all the mice in the rEgmMDH group with 118 cysts totally; and cysts were found in 7 of 9 mice in the PBS control with 35 cysts totally. The mice in the rEgferritin group showed an 84.7% protection but revealed no protection in the rEgmMDH group. The CD4+ T cells were significantly higher in the rEgferritin group than the control, but no statistical difference was found in CD8+ T cells and CD4+/CD8+ ratio between the 2 groups. There was no considerable change in the T cells and ratio in the rEgmMDH group compared to the control. CONCLUSION: The Egrecombinant ferritin can inhibit the growth of Eg while the Egrecombinant mMDH seems promoting its growth in mice.

CD4+CD25+ Treg induction by an HSP60-derived peptide SJMHE1 from Schistosoma japonicum is TLR2 dependent.

Chronic schistosome infection results in the suppression of host immune responses, allowing long-term schistosome survival and restricting pathology. Current theories suggest that Treg play an important role in this regulation. However, the mechanism of Treg induction during schistosome infection is still unknown. The aim of this study was to determine the mechanism behind the induction of CD4(+)CD25(+) T cells by Schistosoma japonicum HSP60 (SjHSP60)-derived peptide SJMHE1 as well as to elucidate the cellular and molecular basis for the induction of CD4(+)CD25(+) T cells during S. japonicum infection. Mice immunized with SJMHE1 or spleen and LN cells from naïve mice pretreated with SJMHE1 in vitro all displayed an increase in CD4(+)CD25(+) T-cell populations. Release of IL-10 and TGF-beta by SJMHE1 stimulation may contribute to suppression. Adoptively transferred SJMHE1-induced CD4(+)CD25(+) T cells inhibited delayed-type hypersensitivity in BALB/c mice. Additionally, SJMHE1-treated APC were tolerogenic and induced CD4(+) cells to differentiate into suppressive CD4(+)CD25(+) Treg. Furthermore, our data support a role for TLR2 in SJMHE1-mediated CD4(+)CD25(+) Treg induction. These findings provide the basis for a more complete understanding of the S. japonicum-host interactions that contribute to host homeostatic mechanisms, preventing an excessive immune response.