The Acute Inflammatory Potential of Particles From the Echinococcus granulosus Laminated Layer Is Moderated by Its Calcium Inositol Hexakisphosphate Component.

Cystic echinococcosis is caused by the tissue-dwelling larva (hydatid) of Echinococcus granulosus sensu lato. A salient feature is that this larva is protected by the acellular laminated layer (LL). As the parasite grows, the LL sheds abundant particles that can accumulate in the parasite's vicinity. The potential of LL particles to induce inflammation in vivo has not been specifically analysed. It is not known how each of its two major components, namely highly glycosylated mucins and calcium inositol hexakisphosphate (InsP6) deposits, impacts inflammation induced by the LL as a whole. In this work, we show that LL particles injected intraperitoneally cause infiltration of eosinophils, neutrophils and monocytes/macrophages as well as the disappearance of resident (large peritoneal) macrophages. Strikingly, the absence of calcium InsP6 enhanced the recruitment of all the inflammatory cell types analysed. In contrast, oxidation of the mucin carbohydrates caused decreased recruitment of neutrophils. The carbohydrate-oxidised particles caused cell influx nonetheless, which may be explained by possible receptor-independent effects of LL particles on innate immune cells, as suggested by previous works from our group. In summary, LL particles can induce acute inflammatory cell recruitment partly dependent on its mucin glycans, and this recruitment is attenuated by the calcium InsP6 component.

Hydatid fluid from Echinococcus granulosus induces autophagy in dendritic cells and promotes polyfunctional T-cell responses.

Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. Echinococcus granulosus is a parasitic tapeworm that causes cystic echinococcosis in animals and humans. The hydatid fluid released by the parasite is known to contain various immunomodulatory components that manipulate host´s defense mechanism. In this study, we focused on understanding the effect of hydatid fluid on dendritic cells and its impact on autophagy induction and subsequent T cell responses. Initially, we observed a marked downregulation of two C-type lectin receptors in the cell membrane, CLEC9A and CD205 and an increase in lysosomal activity, suggesting an active cellular response to hydatid fluid. Subsequently, we visualized ultrastructural changes in stimulated dendritic cells, revealing the presence of macroautophagy, characterized by the formation of autophagosomes, phagophores, and phagolysosomes in the cell cytoplasm. To further elucidate the underlying molecular mechanisms involved in hydatid fluid-induced autophagy, we analyzed the expression of autophagy-related genes in stimulated dendritic cells. Our results demonstrated a significant upregulation of beclin-1, atg16l1 and atg12, indicating the induction of autophagy machinery in response to hydatid fluid exposure. Additionally, using confocal microscopy, we observed an accumulation of LC3 in dendritic cell autophagosomes, confirming the activation of this catabolic pathway associated with antigen presentation. Finally, to evaluate the functional consequences of hydatid fluid-induced autophagy in DCs, we evaluated cytokine transcription in the splenocytes. Remarkably, a robust polyfunctional T cell response, with inhibition of Th2 profile, is characterized by an increase in the expression of il-6, il-10, il-12, tnf-α, ifn-γ and tgf-ß genes. These findings suggest that hydatid fluid-induced autophagy in dendritic cells plays a crucial role in shaping the subsequent T cell responses, which is important for a better understanding of host-parasite interactions in cystic echinococcosis.

Comparative analysis of host immune responses to Hydatid cyst in human and ovine hepatic cystic Echinococcosis.

BACKGROUND: Hydatid disease is caused by the larval stages of the canine tapeworm Echinococcus granulosus. It is one of the most critical helminthic diseases, representing worldwide public health and socio-economic concern. AIM: This study aimed to investigate the expression of apoptosis and immune response within hepatic tissues of humans and sheep infected with the Hydatid cyst. METHODS: Paraffin-embedded tissue was prepared from each tissue sample and used for histopathological examination by Haematoxylin- Eosin. Also, toluidine blue staining was used for mast cell detection, while an immunohistochemical study was performed to assess CD3 T lymphocytes, CD4 helper T lymphocytes, CD8 cytotoxic T lymphocytes, CD20 memory B lymphocytes, CD68 macrophage, and caspase-3 antibodies. RESULTS: The histological examination revealed significant changes, including the infiltration of inflammatory cells, predominantly lymphocytes with scattered giant cells, necrotic hepatic tissue, and fibrosis. Toluidine blue stain revealed a higher number of mast cells (5 cells/field) in humans compared to sheep (3.6 cells/field). The immunohistochemical analysis confirmed that the CD3 were the most predominant inflammatory cell in the hepatic tissue of humans (intensive 70%), and sheep (moderate 38.47%). Caspase-3 was observed in all samples in different grades and mostly in human liver tissue. CONCLUSION: This data could aid in recognizing immunological markers for differentiating disease progression, as well as enhance the understanding of local immune responses to cystic Echinococcosis (CE). The findings could provide preliminary data for future studies on immune responses associated with Hydatid cysts.

Immunization with a Mu-class glutathione transferase from Echinococcus granulosus induces efficient antibody responses and confers long-term protection against secondary cystic echinococcosis.

Cystic echinococcosis, a zoonosis caused by cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) genetic complex, affects humans and diverse livestock species. Although a veterinary vaccine exhibiting high levels of antibody-mediated protection has successfully reached the market, the large genetic diversity among parasite isolates and their particular host preferences, makes still necessary the search for novel vaccine candidates. Glutathione transferases (GSTs) constitute attractive targets for immunoprophylaxis due to their outstanding relevance in helminth detoxification processes, against both exogenous and endogenous stressors. Among the six GSTs known to be expressed in E. granulosus s.l., EgGST1 (Mu-class), EgGST2 (Sigma-class), and EgGST3 (a still non-classifiable isoenzyme), show the highest proteomic expression. Therefore, their recombinant forms -rEgGST1, rEgGST2 and rEgGST3- were herein analyzed regarding their potential to induce long-term antiparasite protection in mice. Only immunization with rEgGST1 induced long-lasting protection; and accordingly, rEgGST1-specific antibodies enhanced the parasite killing through both the classical activation of the host complement system and the antibody-dependent cellular cytotoxicity by macrophages. These results support further testing of rEgGST1 as a vaccine candidate in diverse hosts due to the broad expression of EgGST1 in different parasite stages and tissues.

Effects of annexin B18 from Echinococcus granulosus sensu lato on mouse macrophages.

Cystic echinococcosis (CE) is a zoonotic disease, caused by Echinococcus granulosus sensu lato (E. granulosus s. l.), which posed significant public health concern globally. E. granulosus s. l. annexin B18 (EgANXB18) acts as a secretory protein, exerting a crucial influence in mediating host-parasite interactions. Recombinant annexin B18 (rEgANXB18) was expressed by Escherichia coli and the immunoreactivity was assessed by western blotting. The binding affinity between rEgANXB18 and total protein of RAW264.7 cells was assessed by ELISA. The impact of rEgANXB18 on the metabolic activity of RAW264.7 cells was assayed by Cell Counting Kit-8 assay. The mRNA levels of polarization markers (inducible nitrous oxide synthase (iNOS) and arginase 1 (Arg1)) and key cellular factors (IL-1ß，IL-6，IL-10 and TNFα) were evaluated by qRT-PCR. rEgANXB18 was successfully expressed and recognized by E. granulosus s.l. infected canine sera, as well as could bind to the total protein of RAW264.7 cells. Additionally, rEgANXB18 could promote metabolic activity at 5, 10, 20, and 40 µg/mL while no significant impact on metabolic activity was observed at 80 µg/mL. Co-culture RAW264.7 cells with rEgANXB18 resulted in significantly upregulation of the transcript levels of polarization markers iNOS and Arg1. Moreover, rEgANXB18 significantly upregulated the transcript levels of IL-1ß, IL-6, TNFα, and IL-10, while dose-effect relationship was observed in IL-1ß, IL-6, and IL-10. Our results indicated that EgANXB18 showed the potential to regulate immune response of macrophages by shifting the cell polarization and cytokine profile, thereby promoting the parasitism of CE.

Echinococcus granulosus Protoscoleces-Derived Exosome-like Vesicles and Egr-miR-277a-3p Promote Dendritic Cell Maturation and Differentiation.

Cystic echinococcosis, a major parasitic disease caused by Echinococcus granulosus, seriously threatens human health. The excretory-secretory (ES) products of E. granulosus can induce immune tolerance in dendritic cells (DCs) to downregulate the host's immune response; however, the effect of exosomes in the ES products on the DCs has remained unclear. This study showed that E. granulosus protoscoleces-derived exosome-like vesicles (PSC-ELVs) could be internalized by bone marrow-derived dendritic cells (BMDCs), allowing for the delivery of the parasite microRNAs to the BMDCs. Moreover, PSC-ELVs induced BMDCs to produce the proinflammatory cytokinesinterleukin (IL)-6, IL-12, IL-ß, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ). PSC-ELVs also upregulated the BMDCs surface marker major histocompatibility complex class II (MHC II), as well as costimulatory molecules CD40, CD80, and CD86. PSC-ELV-derived egr-miR-277a-3p upregulated the IL-6, IL-12, and TNF-α mRNA levels in BMDCs. Moreover, egr-miR-277a-3p directly targeted Nfkb1 (encoding nuclear factor kappa B 1) to significantly suppress the mRNA and protein levels of NF-κB1 in BMDCs, while the expression of NF-κB p65 significantly increased, suggesting that egr-miR-277a-3p induces the production of proinflammatory cytokines by the modification of the NF-kB p65/p50 ratio in BMDCs. These results demonstrated that PSC-ELVs and egr-miR-277a-3p might enhance DCs maturation and differentiation in a cross-species manner, which in turn may modulate the host immune responses and offer a new approach to echinococcosis prevention and treatment.

The Recombinant Eg.P29-Mediated miR-126a-5p Promotes the Differentiation of Mouse Naive CD4+ T Cells via DLK1-Mediated Notch1 Signal Pathway.

Cystic echinococcosis (CE) is a zoonotic parasitic disease spread worldwide caused by Echinococcus granulosus (Eg), which sometimes causes serious damage; however, in many cases, people are not aware that they are infected. A number of recombinant vaccines based on Eg are used to evaluate their effectiveness against the infection. Our previous report showed that recombinant Eg.P29 (rEg.P29) has a marvelous immunoprotection and can induce Th1 immune response. Furthermore, data of miRNA microarray in mice spleen CD4+ T cells showed that miR-126a-5p was significantly elevated 1 week after immunization by using rEg.P29. Therefore, in this perspective, we discussed the role of miR-126a-5p in the differentiation of naive CD4+ T cells into Th1/Th2 under rEg.P29 immunization and determined the mechanisms associated with delta-like 1 homolog (DLK1) and Notch1 signaling pathway. One week after P29 immunization of mice, we found that miR-126a-5p was significantly increased and DLK1 expression was decreased, while Notch1 pathway activation was enhanced and Th1 response was significantly stronger. The identical conclusion was obtained by overexpression of mmu-miR-126a-5p in primary naive CD4+ T cells in mice. Intriguingly, mmu-miR-126a-5p was significantly raised in serum from mice infected with protoscolex in the early stages of infection and markedly declined in the late stages of infection, while has-miR-126-5p expression was dramatically reduced in serum from CE patients. Taken together, we show that miR-126a-5p functions as a positive regulator of Notch1-mediated differentiation of CD4+ T cells into Th1 through downregulating DLK1 in vivo and in vitro. Hsa-miR-126-5p is potentially a very promising diagnostic biomarker for CE.

Determination of macrophage types by immunohistochemical methods in the local immune response to liver hydatid cysts in sheep.

Cystic echinococcosis is a zoonotic parasitic disease caused by Echinococcus granulosus. The main hosts in the life cycle of this parasite are dogs and other carnivores; The intermediate hosts are human, sheep, goat, cattle, pig, buffalo, horse and camel. The parasite damages the tissue by forming lesions in the form of fluid-filled cysts in the liver. These lesions are bounded by a layer of local inflammatory cells formed by the host. In the layer formed by this inflammatory response, there are lymphocytes, neutrophils and eosinophil leukocytes, including macrophages. Samples taken from sheep with hydatid cysts in their livers were followed for pathological analysis, and then histopathological and immunohistochemical examinations were performed. After histopathological examinations, the types of macrophages involved in the local immune response against cysts in the liver were determined by immunohistochemical methods using anti-INOS and anti-IL-10 antibodies. INOS and IL-10 immunopositivity were detected in all samples. Statistically, no significant difference was observed between these immunopositivity. This showed that both macrophage types are involved in the local immune response to hydatid cyst, and that Th1 and Th2 immune response stimulation continues together. It was concluded that in future studies that will be planned and experimentally, it will be possible to reveal more clearly how these macrophage types take part in the local immune response.

High Expression of Tim-3 in Alveolar Echinococcosis Mediates Depletion of CD8+ T Cell Function.

OBJECTIVE: CD8+ T cells can participate in immune action by secreting various cytokines, which have a killing effect on certain viruses, tumor cells, and other antigenic substances. However, in studies such as chronic viral infections and some parasitic infections, CD8+ T lymphocyte showed functional depletion, and its immune dysfunction was an important reason for the persistence of infection. Tim-3 has been shown to be a negative regulator of CD8+ T cell function, causing depletion of CD8+ T cells in cancer and chronic infection. However, the relationship between Tim-3 and CD8+ T cells in Echinococcus multilocularis infection is not clear. METHODS: In this study, we analyzed peripheral blood CD8+ T cells from 62 alveolar echinococcosis (AE) patients and 30 healthy controls. RESULTS: Compared with the healthy control group, the proportion of CD8+ T cells in the peripheral blood of AE patients increased significantly, while the levels of perforin, granzyme B and IFN-γ in peripheral blood CD8+ T cell related factors of metabolically active alveolar echinococcosis (MAAE) patients decreased significantly. Later detection revealed that the expression of Tim-3 on CD8+ T cells in the peripheral blood of MAAE patients was significantly higher than that of metabolically inactive alveolar echinococcosis (MIAE) patients and healthy controls. The expression levels of function-related factors perforin, granzyme B and IFN-γ in CD8+ Tim-3+ T cell were significantly lower in the CD8+Tim-3- T cells of AE patients. In vitro, the secretion of CD8+ T cell-associated factors was significantly restored by inhibiting Tim-3 expression. CONCLUSION: Therefore, the depletion of CD8+ T lymphocyte in patients with alveolar echinococcosis disease is considered to be related to the high expression of Tim-3 on the surface.

Synergism therapeutic and immunoregulatory effects of Albendazole + rAd-mIL-28B against Echinococcosis in experiment-infected mice with protoscoleces.

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×107 PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P < 0.05), especially in Albendazole (ABZ) + rAd-mIL-28B group (5.8 ± 1.4 g) (P < 0.01). We also observed the severe damage of the germinal layer and the laminated layer of cysts after treatment. rAd-mIL-28B group showed a prominent increase in the level of Th1 type cytokines (such as IFN-γ, IL-2 and TNF-α). Meanwhile, the frequency of Foxp3+ T cells was decreased in the rAd-mIL-28B group (4.83 ± 0.81%) and ABZ + rAd-mIL-28B group (4.60 ± 0.51%), comparing with the untreated group (8.13 ± 2.60%) (P < 0.05). In addition, compared with the untreated control (122.14 ± 81.09 pg/ml), the level of IFN-γ significantly increased in peritoneal fluid in the rAd-mIL-28B group (628.87 ± 467.16 pg/ml) (P < 0.05) and ABZ + rAd-mIL-28B group (999.76 ± 587.60 pg/ml) (P < 0.001). Taken together, it suggested that ABZ + IL-28B may be a potential therapeutic agent against CE.

Molecular characterization and immune protection of the 3-hydroxyacyl-CoA dehydrogenase gene in Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis (CE) is a serious parasitic zoonosis caused by the larvae of the tapeworm Echinococcus granulosus. The development of an effective vaccine is one of the most promising strategies for controlling CE. METHODS: The E. granulosus 3-hydroxyacyl-CoA dehydrogenase (EgHCDH) gene was cloned and expressed in Escherichia coli. The distribution of EgHCDH in protoscoleces (PSCs) and adult worms was analyzed using immunofluorescence. The transcript levels of EgHCDH in PSCs and adult worms were analyzed using quantitative real-time reverse transcription PCR (RT-qPCR). The immune protective effects of the rEgHCDH were evaluated. RESULTS: The 924-bp open reading frame sequence of EgHCDH, which encodes a protein of approximately 34 kDa, was obtained. RT-qPCR analysis revealed that EgHCDH was expressed in both the PSCs and adult worms of E. granulosus. Immunofluorescence analysis showed that EgHCDH was mainly localized in the tegument of PSCs and adult worms. Western blot analysis showed that the recombinant protein was recognized by E. granulosus-infected dog sera. Animal challenge experiments demonstrated that dogs immunized with recombinant (r)EgHCDH had significantly higher serum IgG, interferon gamma and interleukin-4 concentrations than the phosphate-buffered saline (PBS) control group. The rEgHCDH vaccine was able to significantly reduce the number of E. granulosus and inhibit the segmental development of E. granulosus compared to the PBS control group. CONCLUSIONS: The results suggest that rEgHCDH can induce partial immune protection against infection with E. granulosus and could be an effective candidate for the development of new vaccines.

Design of a Novel Multi-Epitope Vaccine Against Echinococcus granulosus in Immunoinformatics.

All the time, echinococcosis is a global zoonotic disease which seriously endangers public health all over the world. In order to speed up the development process of anti-Echinococcus granulosus vaccine, at the same time, it can also save economic cost. In this study, immunoinformatics tools and molecular docking methods were used to predict and screen the antigen epitopes of Echinococcus granulosus, to design a multi-epitope vaccine containing B- and T-cell epitopes. The multi-epitope vaccine could activate B lymphocytes to produce specific antibodies theoretically, which could protect the human body against Echinococcus granulosus infection. It also could activate T lymphocytes and clear the infected parasites in the body. In this study, four CD8+ T-cell epitopes, three CD4+ T-cell epitopes and four B-cell epitopes of Protein EgTeg were identified by immunoinformatics methods. Meanwhile, three CD8+ T-cell epitopes, two CD4+ T-cell epitopes and four B-cell epitopes of Protein EgFABP1 were identified. We constructed the multi-epitope vaccine using linker proteins. The study based on the traditional methods of antigen epitope prediction, further optimized the prediction results combined with molecular docking technology and improved the precision and accuracy of the results. Finally, in vivo and in vitro experiments had verified that the vaccine designed in this study had good antigenicity and immunogenicity.

Evaluation of inflammatory parameters in patients with hepatic hydatid disease.

BACKGROUND: To the best of our knowledge, the association of inflammatory parameters with hepatic hydatid disease (HD) has not been investigated in a single study. We aimed to evaluate the potential value of inflammatory indices in this disorder. METHODS: The retrospective study including 114 patients was performed from January 2016 to November 2019. Clinical characteristics and laboratory data for all participants were collected and analysed. The levels of inflammatory parameters were compared in the patient and control group, the predictive value of these inflammatory parameters was assessed by the logistic regression analysis and receiver operating characteristic curve, and differences between pre- and post-surgical operations were compared by pair tests. RESULTS: Significantly higher levels of platelet distribution width (PDW), eosinophil percentage (EOS %), neutrophil to lymphocyte ratio (NLR), gamma-glutamyl transpeptidase to platelet ratio (GPR) and alkaline phosphatase to platelet ratio (APPR) and lower levels of platelet (PLT) and prognostic nutritional index (PNI) were observed in patients than in controls. Multivariate analyses showed that hydatid could induce the abnormal levels of these parameters, of which APPR and PNI had more obvious changes as compared to other parameters. The levels of PDW and APPR significantly decreased after surgical treatment. CONCLUSIONS: Inflammatory parameters closely associated with the hepatic HD could be used in the evaluation of treatment as assistant indexes.KEY MESSAGEHydatid disease (HD) seriously endangers public health and economic development.Inflammatory parameters that are readily available and acceptable in routine clinical practice could be closely associated with HD.Inflammatory parameters could be used in the evaluation of disease development by combing with histological and radiological results in future studies.

Microcyst fluid promotes the migration and invasion of fibroblasts in the adventitial layer of alveolar echinococcosis.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis (E. multilocularis), characterized by lesions composed of an aggregate of microcysts embedded in a granulomatous host's reaction. The periphery of parasite granulomas often additionally displays fibrotic reactions of varying intensity, in which E. multilocularis microenvironment fibroblasts (EMFs) laid down collagen. However, the regulation of EMFs by the infiltration of E. multilocularis microcyst fluid (MF) into granulomas remains poorly defined. This study aimed to investigate the effect of MF on migration and invasion of primary isolated EMFs cells. A mouse model of secondary infection with AE was established, and the model construction was evaluated by HE staining. EMFs were cultured in primary by tissue block adherency method. The isolated cells were identified by qPCR, immunofluorescence and Western blot. Then CCK-8 assay, cell migration/invasion assay and flow cytometry were performed to detect the effects of MF on the proliferation, migration, invasion and cell cycle of EMFs, respectively. The expressions of MMP2 and MMP9 at mRNA and protein levels in EMFs were detected by RT-qPCR and Western blot. The effect of PI3K-Akt signal transduction pathway on regulating the expression of MMPs expression was assessed by Western blot. As indicated from the results, EMFs were successfully isolated from the E. multilocularis microenvironment and identified as myofibroblasts. MF significantly facilitated the proliferation and cell cycle progression of EMFs. In addition, MF significantly improved the migration and invasion of EMFs. MF was further confirmed to up-regulate mRNA and protein expressions of MMP2 and MMP9 in EMFs, which was related to the activation of the PI3K-Akt signaling pathway. The present study demonstrates that MF can promote the migration and invasion of EMFs cells significantly, which might be via activating PI3K-Akt signaling pathway.

Modulation of the mTOR pathway plays a central role in dendritic cell functions after Echinococcus granulosus antigen recognition.

Immune evasion is a hallmark of persistent echinococcal infection, comprising modulation of innate immune cells and antigen-specific T cell responses. However, recognition of Echinococcus granulosus by dendritic cells (DCs) is a key determinant of the host's response to this parasite. Given that mTOR signaling pathway has been described as a regulator linking metabolism and immune function in DCs, we reported for the first time in these cells, global translation levels, antigen uptake, phenotype, cytokine transcriptional levels, and splenocyte priming activity upon recognition of the hydatid fluid (HF) and the highly glycosylated laminar layer (LL). We found that LL induced a slight up-regulation of CD86 and MHC II in DCs and also stimulated the production of IL-6 and TNF-α. By contrast, HF did not increase the expression of any co-stimulatory molecules, but also down-modulated CD40 and stimulated the expression of the anti-inflammatory cytokine IL-10. Both parasitic antigens promoted protein synthesis through mTOR activation. The use of rapamycin decreased the expression of the cytokines tested, empowered the down-modulation of CD40 and also reduced splenocyte proliferation. Finally, we showed that E. granulosus antigens increase the amounts of LC3-positive structures in DCs which play critical roles in the presentation of these antigens to T cells.

Anti-cancer immunoprotective effects of immunization with hydatid cyst wall antigens in a non-immunogenic and metastatic triple-negative murine mammary carcinoma model.

Cancer vaccines have gained lots of attention as the future of cancer treatment. However, poor immunogenicity of tumor-associated antigens often fails to induce an efficient immune response against the tumor. Strange anti-tumor immune responses at the parasite-infected patients due to cross-reactivity have been reported in various studies. Therefore, parasite antigens with significant immunogenicity and high epitope homology with cancer antigens may activate a strong immune response against cancer cells. Herein, the sera of immunized rabbits with the hydatid cyst wall (HCW) antigens were incubated with 4 T1 mammary carcinoma cells to investigate cross-reactivity between the HCW antigens antisera and surface antigens of the breast cancer cells. Also, the SDS-PAGE profile of HCW antigens was prepared and incubated with the breast cancer patients' sera and considerable reactivity was observed between their sera and a specific band (~27/28 kDa) according to Western blotting analyzes. Then, the protein bands with cross-reactivity with breast cancer patients' sera were utilized for prophylactic immunizations of Balb/c mice. The immunoprotective effect of immunization with the HCW antigens caused significant inhibition of 4 T1 breast tumor growth, decrease of metastasis, and enlargement of the tumor-bearing mice survival time in comparison with PBS and pure immune adjuvant injected groups. Mass spectrometry analysis showed that the ~ 27/28 kDa band has numbers of proteins/polypeptides with a high degree of homology with cancer cells antigens which can be the reason for this cross-reactivity and anti-tumor immune response. Taking together, immunization with HCW antigens would be a promising approach in cancer immunotherapy after further investigations.

The Relationship Between Preoperative Systemic Immune Inflammation Index and Prognostic Nutritional Index and the Prognosis of Patients With Alveolar Hydatid Disease.

Background: To explore the relationship between the preoperative immune inflammation index (SII) and the prognostic nutritional index (PNI) and the overall survival rate (OS) of patients with alveolar hydatid disease. Methods: The clinical data of patients with hepatic alveolar echinococcosis treated by surgery in the Department of Hepatobiliary and Pancreatic Surgery, Affiliated Hospital of Qinghai University from January 2015 to January 2019 were analyzed retrospectively, and the SII, PNI, PLR and NLR were calculated. Spearman correlation analysis was utilized to analyze the correlation among SII, PNI, PLR and NLR. Receiver operating characteristic curve (ROC) was utilized to determine the best intercept values of SII, PNI, PLR and NLR, and Chi-square test was used to evaluate the relationship between SII, PNI and various clinicopathological features in patients with hepatic alveolar echinococcosis. The kaplan-Meier method was used to draw survival curves and analyze the relationship between them and the total survival time of patients. A cox regression model was used to analyze the relationship between SII, PNI and the prognosis of patients with hepatic alveolar echinococcosis. Finally, ROC curve was used to estimate the predictive efficacy of SII, PNI and COSII-PNI for the prognosis of patients with hepatic alveolar echinococcosis. Results: A total of 242 patients were included, including 96 males and 146 females, aged 11.0-67.0 (36.6 ± 11.7) years. The values of SII, PNI, PLR and NLR are calculated, and the best truncation values of SII, PNI, PLR and NLR are given in ROC curve. The kaplan-Meier survival curve was used to analyze the relationship between SII, PNI, PLR, NLR and the overall survival time of patients with hepatic alveolar echinococcosis. The results showed that the median follow-up time was 45 months (95%CI: 39.484-50.516), and the average survival time was 49 months (95%CI: 47.300-51.931), which was low p<0.001); The 5-year OS rate of low PNI was significantly lower than that of high PNI group (37.7% vs 71.6%; p<0.001); The 5-year OS rate in low PLR group was significantly higher than that in high PLR group (70.4% vs 24.3%; p<0.001); The 5-year OS rate in low NLR group was significantly higher than that in high NLR group (67.2% vs 28.8%; p<0.001). Cox unifoliate analysis showed that SII, PNI, PLR and NLR were important prognostic factors related to OS. Cox multivariate analysis showed that SII(HR=4.678, 95% CI: 2.581-8.480, P<0.001) and PNI(HR=0.530, 95%CI: 0.305-0.920, P<0.05) were identified as independent risk indicators of OS, while NL was identified as independent risk indicators of OS ROC curve analysis showed that AUC of SII, PNI, PLR, NLR and COSII-PNI were 0.670(95%CI: 0.601-0.738), 0.638(95%CI: 0.561-0.716) and 0.618(95% CI: 0.541-0.694), respectively COSII-PNI is superior to SII and PNI in evaluating prognosis (P < 0.05). Conclusions: SII and PNI can be regarded as independent risk factors reflecting the prognosis of patients with hepatic alveolar echinococcosis. The lower SII and the higher PNI before operation, the better the prognosis of patients, and the combined application of SII and PNI before operation can improve the accuracy of prediction.

Enzymatic characteristics and preventive effect of leucine aminopeptidase against Echinococcus multilocularis.

Alveolar echinococcosis, a parasitic zoonotic disease caused by the larval stage of Echinococcus multilocularis infection, is a global epidemic in Eurasia and North America. Leucine aminopeptidase (LAP) of the M17 peptidase family could act on an ideal target antigen in diagnosis and prevention of parasitic diseases (schistosomiasis, malaria, fascioliasis) because of its good immunogenicity. In this study, the bioinformatic and enzymatic characterizations of recombinant Echinococcus multilocularis LAP (rEm-LAP) were evaluated. A prokaryotic expression system for rEm-LAP protein was established and its immunogenicity and preventive efficacy were demonstrated in a BALB/c mice model. This is the first report about the LAP of Echinococcus multilocularis and with a 57.4 KD purified rEm-LAP protein successfully expressed by pCzn1-LAP in Escherichia coli BL-21 cells. Enzymatic analysis results showed optimal rEm-LAP activity at pH 9. Serum indirect ELISA demonstrated that rEm-LAP could induce a Th1 and Th2 mixed-type immunological response and produce high levels of IgG, IgG1, IgG2a, IgM, and IgA. Furthermore, serum IFN-γ and IL-4 secretion were increased compared with the control groups. Finally, vaccination with rEm-LAP significantly decreased both the number and size of the cysts in Echinococcus multilocularis metacestode infected mice model. The current study provides evidence that rEm-LAP could be a potential vaccine antigen of Echinococcus multilocularis.

Involvement of TIGIT in Natural Killer Cell Exhaustion and Immune Escape in Patients and Mouse Model With Liver Echinococcus multilocularis Infection.

BACKGROUND AND AIMS: Alveolar echinococcosis (AE) is a lethal helminthic liver disease caused by persistent infection with Echinococcus multilocularis. Although more attention has been paid to the immunotolerance of T cells caused by E. multilocularis infection, the role of natural killer (NK) cell, a critical player in liver immunity, is seldom studied. APPROACH AND RESULTS: Here, we observed that NK cells from the blood and closed liver tissue (CLT) of AE patients expressed a higher level of inhibitory receptor TIGIT and were functionally exhausted with a lower expression of granzyme B, perforin, interferon-gamma (IFN-γ), and TNF-α. Addition of anti-TIGIT (T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain) monoclonal antibody into AE patients' peripheral blood mononuclear cell culture significantly enhanced the synthesis of IFN-γ and TNF-α by NK cells, indicating the reversion of exhausted NK cells by TIGIT blockade. In the mouse model of E. multilocularis infection, liver and splenic TIGIT+ NK cells progressively increased dependent of infection dosage and timing and were less activated and less degranulated with lower cytokine secretion. Furthermore, TIGIT deficiency or blockade in vivo inhibited liver metacestode growth, reduced liver injury, and increased the level of IFN-γ produced by liver NK cells. Interestingly, NK cells from mice with persistent chronic infection expressed a higher level of TIGIT compared to self-healing mice. To look further into the mechanisms, more regulatory CD56bright and murine CD49a+ NK cells with higher TIGIT expression existed in livers of AE patients and mice infected with E. multilocularis, respectively. They coexpressed higher surface programmed death ligand 1 and secreted more IL-10, two strong inducers to mediate the functional exhaustion of NK cells. CONCLUSIONS: Our results indicate that inhibitory receptor TIGIT is involved in NK cell exhaustion and immune escape from E. multilocularis infection.