Multilocularis Infection Research Articles

Therapeutic effect on Alveolar echinococcosis by targeting EM-Leucine aminopeptidase.

Alveolar echinococcosis (AE) is a parasitic disease caused by E. multilocularis metacestodes and it is highly prevalent in the northern hemisphere. We have previously found that vaccination with E. multilocularis Leucine aminopeptidase (EM-LAP) induced specific immune response and had an inhibiting effect on the parasites. In this study, the therapeutic effect of recombinant EM-LAP (rEM-LAP) on AE was evaluated and verified using Ubenimex, a broad-spectrum inhibitor of LAP. The results reveal that rEM-LAP could inhibit cyst growth and invasion and induce specific immunity response in BALB/c mice infected with E. multilocularis protoscoleces. The ultrasonic, MRI, and morphological results show that treatment with rEM-LAP inhibits E. multilocularis infection and reduces cyst weight, number, fibrosis and invasion. The same effect is observed for the treatment with Ubenimex by inhibiting LAP activity. The indirect ELISA shows that rEM-LAP could induce specific immunity response and produce high levels of IgG, IgG1, IgG2a, IgM, and IgA, and the serum levels of IFN-γ and IL-4 are significantly increased compared to the control groups, indicating that treatment with rEM-LAP leads to a Th1 and Th2 mixed-type immune response. This study suggests that EM-LAP could be a potential therapeutic target of E. multilocularis infection.

Immunization with EmCRT-Induced Protective Immunity against Echinococcus multilocularis Infection in BALB/c Mice

Alveolar echinococcosis (AE) is a severe parasitic zoonosis caused by the larval stage of Echinococcus multilocularis. The identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Echinococcus infection. Here, we identified that E. multilocularis calreticulin (EmCRT), a ubiquitous protein with a Ca2+-binding ability, could be recognized by the sera of mice infected with E. multilocularis. The native EmCRT was expressed on the surface of E. multilocularis larvae as well as in the secreted products of metacestode vesicles and protoscoleces (PSCs). The coding DNA for EmCRT was cloned from the mRNA of the E. multilocularis metacestode vesicles and a recombinant EmCRT protein (rEmCRT) was expressed in E. coli. Mice immunized with soluble rEmCRT formulated with Freund’s adjuvant (FA) produced a 43.16% larval vesicle weight reduction against the challenge of E. multilocularis PSCs compared to those that received the PBS control associated with a high titer of IgG, IgG1 and IgG2a antibody responses as well as high levels of Th1 cytokines (IFN-γ and IL-2) and Th2 cytokines (IL-4, IL-5 and IL-10), produced by splenocytes. Our results suggest that EmCRT is an immunodominant protein secreted by E. multilocularis larvae and a vaccine candidate that induces partial protective immunity in vaccinated mice against Echinococcus infection.

Monocyte-derived macrophages: The supplements of hepatic macrophage in Echinococcus multilocularis infected mice.

BackgroundAlveolar echinococcosis is a potentially lethal zoonosis caused by the cestode Echinococcus multilocularis. This study is to investigate the dynamic changes of monocytes, macrophages, and related cytokines in animal models of persistent infection of E. multilocularis.MethodsAn infection model was established by intraperitoneal injection of a protoscolex suspension. The pathological changes of liver were observed by HE staining. The percentage of Ly6Chi and Ly6Clo Monocytes in peripheral blood was detected by flow cytometry. The distribution and expression of CX3CL1, CX3CR1, iNOS, CD163, and CD11b in the liver were detected by immunohistochemistry. The mRNA expression of tumor necrosis factor‐α (TNF‐α) and Arg1 in the liver was detected by quantitative reverse transcription polymerase chain reaction. The expression of INF‐γ, interleukin‐17 (IL‐17), IL‐4, and IL‐10 in peripheral blood was detected by enzyme‐linked immunosorbent assay.ResultsHematoxylin‐eosin(HE) staining showed that significant lesions appeared in the later stages of infection in the liver. The proportion of Ly6Chi monocytes in the peripheral blood of the experimental group mice decreased after a brief rise, Ly6Clo monocytes decreased first and then increased. The expression of CX3CL1, CX3CR1, CD11b, CD163, and iNOS in the mice liver of the experimental group was increased. The expression level of TNF‐α and Arg1 mRNA in the liver of the experimental group mice increased. The expression level of IFN‐γ, IL‐17, IL‐4, and IL‐10 increased with the duration of infection.ConclusionsMonocytes as a supplement to hepatic macrophage, monocytes and kupffer cells may both participate in Th1 and Th2 immune responses by differentiating into M1 or M2 at different stages of E. multilocularis infection.

Effects of Echinococcus multilocularis metacestodes infection and drug treatment on the activities of biotransformation enzymes in mouse liver.

The changes of biotransformation enzymes will substantially affect the host's ability to metabolize drugs and other xenobiotic compounds. In order to further elucidate this process and promote the development in treatment of echinococcosis, we investigated the effects of Echinococcus multilocularis infection and drug treatment on biotransformation enzymes in mouse liver. In microsomal and cytosolic fractions, from the six activities assayed, significant decrease of glutathione S-transferases (GST) activity and significant increase of 7-pentoxyresorufin (PROD) and NADPH-cytochrome P450 reductase (CPR) activity were observed in the mice infected withE. multilocularismetacestodes. In addition, after six weeks treatment of albendazole inE. multilocularisinfected mice, significant decreased GST activity and significant increase of 7- ethoxyresorufin (EROD), PROD, and particularly 3-fold higher 7-methoxyresorufin (MROD) activity were observed. The 3-bromopyruvate treated mice only exhibited significantly lower GST activity. Our results demonstrate thatE. multilocularismetacestodes infection can affect the activities of main hepatic biotransformation enzymes and such alterations of activity may further affect the hepatic biotransformation of xenobiotics. Moreover, albendazole and 3-bromopyruvate, the promising potential drug againstEchinococcus, affected different hepatic biotransformation enzymes and may affect their metabolism. The findings will help to develop rational treatments with less side effects and promote the development of more efficient treatments againstE. multilocularis.

HIV protease inhibitor nelfinavir is a potent drug candidate against echinococcosis by targeting Ddi1-like protein.

SummaryBackgroundAlveolar echinococcosis (AE), which is caused by larval Echinococcus multilocularis, is one of the world's most dangerous neglected diseases. Currently, no fully effective treatments are available to cure this disease.MethodsIn vitro protoscolicidal assay along with in vivo murine models was applied in repurposing drugs against AE. Genome-wide identification and homology-based modeling were used for predicting drug targets. RNAi, enzyme assay, and RNA-Seq analyses were utilized for investigating the roles in parasite survival and validations for the drug target.FindingsWe identified nelfinavir as the most effective HIV protease inhibitor against larval E. multilocularis. Once-daily oral administration of nelfinavir for 28 days resulted in a remarkable reduction in parasite infection in either immune-competent or immunocompromised mice. E. multilocularis DNA damage-inducible 1 protein (EmuDdi1) is predicted as a target candidate for nelfinavir. We proved that EmuDdi1 is essential for parasite survival and protein excretion and acts as a functionally active protease for this helminth. We found nelfinavir is able to inhibit the proteolytic activity of recombinant EmuDdi1 and block the EmuDdi1-related pathways for protein export. With other evidence of drug efficacy comparison, our results suggest that inhibition of EmuDdi1 is a mechanism by which this HIV proteinase inhibitor mediates its antiparasitic action on echinococcosis.InterpretationThis study demonstrates that nelfinavir is a promising candidate for treating echinococcosis. This drug repurposing study proves that the widely prescribed drug for AIDS treatment is potent in combating E. multilocularis infection and thus provides valuable insights into the development of single-drug therapy for highly prevalent co-infection between HIV and helminth diseases.FundingThis work was supported by the National Natural Science Foundation of China (31802179), the Natural Science Foundation of Gansu Province, China (No. 21JR7RA027), and the State Key Laboratory of Veterinary Etiological Biology (No. SKLVEB2021YQRC01).

Alveolar echinococcosis in nutria (Myocastor coypus), invasive species in Slovenia

The present report describes a case of Echinococcus multilocularis infection in nutria (Myocastor coypus) culled in the central area of Slovenia. Post-mortem exam showed multiple cystic lesions in the liver. Gross examination, as well as parasitological and histopathological examinations, revealed numerous cysts of various sizes, filled with yellow clear fluid and displacing most of the liver parenchyma. The cyst lumina contained numerous protoscolices approximately 100 μm in diameter and calcareous corpuscles. The protoscolices had two visible suckers and a rostellum with birefringent hooks. The lesions were consistent with an E. multilocularis cyst. Molecular analysis confirmed that the nutria was infected with E. multilocularis. To our knowledge, this is the first report of echinococcosis in nutria in Slovenia that presents gross, parasitological, and histological lesions and the result of molecular analysis. Nutrias in Slovenia are dangerous invaders but can also be a relevant bioindicator of the presence of the parasite in the environment.

Efficient delivery of Echinococcus multilocularis miRNAs using chitosan nanoparticles.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease, a great threat to human health due to limited interventions. microRNAs are a type of small non-coding RNA that plays a key role in many diseases and is considered as a potential therapeutic target for control of parasitic diseases. However, naked miRNAs are difficult to enter into cells and are easily degraded in both external and internal environments. Chitosan (CS) has recently been used as a promising vehicle for delivery of nucleic acids. Therefore, we prepared miRNA-bearing CS nanoparticles and investigated the physicochemical properties as well as the delivery efficiency. We found that CS nanoparticles was relatively stable, offered miRNA strong protection from degradation and had low cytotoxicity with no significant effects on cell proliferation and apoptosis. CS nanoparticles were shown to be easily absorbed by cells and have remarkable liver tropism. Furthermore, CS nanoparticles were used to efficiently deliver E. multilocularis miR-4989 in vitro and in vivo and caused a significant reduction in the expression of UBE2N in the liver, a potential target of emu-miR-4989, at both mRNA and protein levels. Our data demonstrate that CS nanoparticles can act as a vehicle for efficient liver-targeted delivery of miRNAs and for development of miRNA-based therapeutics against E. multilocularis infection.

Understanding pathogen-host interplay by expression profiles of lncRNA and mRNA in the liver of Echinococcus multilocularis-infected mice.

Almost all Echinococcus multilocularis (Em) infections occur in the liver of the intermediate host, causing a lethal zoonotic helminthic disease, alveolar echinococcosis (AE). However, the long non-coding RNAs (lncRNAs) expression profiles of the host and the potential regulatory function of lncRNA during Em infection are poorly understood. In this study, the profiles of lncRNAs and mRNAs in the liver of mice at different time points after Em infection were explored by microarray. Thirty-one differentially expressed mRNAs (DEMs) and 68 differentially expressed lncRNAs (DELs) were found continuously dysregulated. These DEMs were notably enriched in “antigen processing and presentation”, “Th1 and Th2 cell differentiation” and “Th17 cell differentiation” pathways. The potential predicted function of DELs revealed that most DELs might influence Th17 cell differentiation and TGF-β/Smad pathway of host by trans-regulating SMAD3, STAT1, and early growth response (EGR) genes. At 30 days post-infection (dpi), up-regulated DEMs were enriched in Toll-like and RIG-I-like receptor signaling pathways, which were validated by qRT-PCR, Western blotting and downstream cytokines detection. Furthermore, flow cytometric analysis and serum levels of the corresponding cytokines confirmed the changes in cell-mediated immunity in host during Em infection that showed Th1 and Th17-type CD4+ T-cells were predominant at the early infection stage whereas Th2-type CD4+ T-cells were significantly higher at the middle/late stage. Collectively, our study revealed the potential regulatory functions of lncRNAs in modulating host Th cell subsets and provide novel clues in understanding the influence of Em infection on host innate and adaptive immune response.

One-tube nested MGB Probe Real-time PCR assay for detection of Echinococcus multilocularis infection in plasma cell free DNA

IntroductionThe main objective of this study was to develop a One-tube nested MGB probe real-time PCR Assay for detecting Echinococcus multilocularis infection in human plasma cell free DNA (cfDNA). MethodscfDNA was extracted from 10 E.m.-infected patients using a NucleoSnap DNA Plasma Kit and characterized by genomic sequencing. We designed nested PCR primers and MGB probe for Echinococcus multilocularis detection. The specificity, sensitivity and reproducibility of this assay were analyzed, and its validity was confirmed in 13 early stage clinical samples. ResultsSeveral Echinococcus multilocularis-specific sequences were detected in the cfDNA of E.m.-infected patients, and CBLO020001206.1 was selected as the candidate sequence. We designed the primers and probe for the one tube nested real-time PCR. No cross-reactions with E.g. were observed. The detection limit was as low as 1 copy for Echinococcus multilocularis. The coefficients of variation were lower than 5% in intra- and inter-assays. 11 out of 13 patients were positive with nested MGB Probe PCR Assay and 3 patients were positive without outer primer in early stage Alveolar Echinococcosis pateints. ConclusionThe one-tube nested MGB probe real-time PCR assay is a simple, rapid, and cost-effective method for detection of Echinococcus multilocularis infection in patients’ Plasma DNA.

Echinococcosis Is Associated with the Increased Prevalence of Intestinal Blastocystis Infection in Tibetans and Host Susceptibility to the Blastocystis in Mice.

Simple SummaryBlastocystis is a neglected enteric pathogen that is highly prevalent in humans and animals worldwide. Studies have reported that Blastocystis infection frequently coexists with other infectious pathogens in humans. However, dual infection by Blastocystis and Echinococcus multilocularis, which causes the severe parasitic disease echinococcosis has not been reported. In this study, the authors investigated the clinical prevalence, risk factors, and genotypes of Blastocystis in Tibetan patients with liver echinococcosis and Tibetan healthy controls from the Qinghai province in China, and also tested whether E. multilocularis infection increases host susceptibility to Blastocystis using a mouse model. The results found a significantly higher prevalence of genetically divergent Blastocystis in Tibetans with liver echinococcosis. E. multilocularis infection in Balb/c mice increased the host susceptibility to Blastocystis and aggravated intestinal pathology with higher disease severity and higher mortality. Taken together, these findings provide new insights into dual infections by Blastocystis and helminths in humans.Blastocystis is a common human intestinal protozoan parasite. Little is known about its prevalence in echinococcosis. This study tested whether Echinococcus multilocularis infection would increase host susceptibility to Blastocystis. A total of 114 fecal samples (68 hydatid disease patients and 46 healthy people) were collected from Tibetans in the Qinghai province in China. The presence of Blastocystis was identified by sequencing of the small subunit (SSU) rRNA gene. Balb/c mice were co-infected with Blastocystis and E. multilocularis and tested for host susceptibility to Blastocystis. The overall Blastocystis prevalence was 12.3%; 16.2% in the patients and 4.4% in healthy people (p < 0.05). Sequence analysis identified three known Blastocystis genotypes, including ST1, ST2, and ST3, and one unknown genotype. Experimental dual infection significantly reduced mouse survival rate (20%), induced more severe signs, and increased intestinal damages with a higher intestinal colonization level of Blastocystis. The mouse model showed that E. multilocularis infection increases host susceptibility to Blastocystis. Our study shows a significantly higher prevalence of Blastocystis in patients with liver echinococcosis and reveals that non-intestinal E. multilocularis infection increases host susceptibility to the Blastocystis. Our results highlight that E. multilocularis infection is associated with Blastocystis. These findings remind us that more attention should be paid to the gut health of the patients with a helminth infection during clinical patient care.

Risk Factors Associated With Echinococcosis in the General Chinese Population: A Meta-Analysis and Systematic Review.

BackgroundEchinococcosis is a severe zoonotic disease that imposes a substantial burden on human life. This meta-analysis aimed to summarize available data on the prevalence of human echinococcosis and identify the key risk factors for echinococcosis in the Chinese general population.MethodsRelevant studies were comprehensively searched in the PubMed, EMBASE, Web of Science, Cochrane, Chinese National Knowledge Infrastructure (CNKI), Chongqing VIP Information (VIP), Wanfang and SinoMed databases until August 22, 2020. A random-effects model was used to estimate the pooled odds ratio (OR) and 95% confidence interval (95% CI). The I2 and Q statistics were calculated to evaluate the heterogeneity, and potential sources of heterogeneity were identified using sensitivity analysis and subgroup analysis. Publication bias was estimated by funnel plots and Egger's test.ResultsA total of 1026 studies were identified through the database search, of which 26 were eligible for this meta-analysis. The pooled prevalence of AE and CE were 2.88% and 5.66%, respectively. Ethnicity (OR = 2.93, 95% CI: 1.81–4.75; I2 = 0), herdsman occupation (OR = 2.66, 95% CI: 2.25–3.14; I2 = 8.0%), not washing hands before meals (OR = 2.40, 95% CI: 1.34–4.28; I2 = 82.8%) and being female (OR = 1.45, 95% CI: 1.26–1.66; I2 = 33.9%) were risk factors for AE. The top five risk factors for CE were ethnicity (OR = 3.18, 95% CI: 1.55–6.52; I2 = 79.2%), nomadism (OR = 2.71, 95% CI: 1.65–4.47; I2 = 55.8%), drinking nonboiled water (OR = 2.47, 95% CI: 1.36–4.47; I2 = 85.7), feeding viscera to dogs (OR = 2.35, 95% CI: 1.89–2.91; I2 = 21.5%), and herdsman occupation (OR = 2.19, 95% CI: 1.67–2.86; I2 = 85.1%).ConclusionsThis study generalized articles that have contributed to our current understanding of the epidemic of human echinococcosis (AE and CE) in China over the years. The results support that the ethnicity and dog-related factors are major risk factors for both CE and AE. The identification of echinococcosis risk factors may aid researchers and policymakers in improving surveillance and preventive measures aimed at reducing Echinococcus granulosus and Echinococcus multilocularis infection in humans.

Progress of researches on infection with two species of Echinococcus causing human diseases in animal hosts and influencing factors

Echinococcosis is a serious zoonotic parasitic disease caused by infections with larval Echinococcus. The life cycle of Echinococcus involves a variety of animal hosts, including hoofed animals and rodents as intermediate hosts and carnivores as definitive hosts. The transmission of human echinococcosis is closely associated with the life cycle of E. granulosus and E. multilocularis among animal hosts in nature. This review summarizes the recent advances in the prevalence and influencing factors of E. granulosus and E. multilocularis infections in animal hosts, so as to provide insights into precision control of echinococcosis.

Assessing the potential for infections of Echinococcus multilocularis in dogs in a hotspot of human alveolar echinococcosis infections in North America.

Echinococcus multilocularis is a zoonotic tapeworm, whose metacestode larval stage is the etiological agent for alveolar echinococcosis in humans and is a parasite of emerging concern according to the World Health Organization which is difficult to diagnose and has a case mortality rate of >90% when left untreated. Echinococcus multilocularis requires two mammalian hosts to complete its lifecycle: wild and domestic canids as definitive hosts, and small mammals (mostly rodents) as intermediate ones. Because of their close relations with humans, domestic dogs have been indicated as a mean of infection to people. Human alveolar echinococcosis has historically been rare in North America, however, since 2013, at least seventeen diagnoses have been confirmed in Alberta, Canada. Because of this unprecedented series of cases, assessing the frequency of infections in dogs in Alberta is key to estimate risk for dog owners and animal health professionals. This study was carried out in Edmonton to determine the frequency of E. multilocularis infection in domestic dogs and potential risk factors. Fecal samples and corresponding behavior risk surveys were collected from 775 dogs in seven urban off-leash parks within Edmonton city limits during the summer of 2020. A quantitative PCR fecal test was used to diagnose E. multilocularis infection. We found a single case of E. multilocularis infection (1/775) and determined that the overall true prevalence was 0.2% (95% CrI: 0.0-0.7%) corrected for detection sensitivity and specificity. Overall, these findings confirm the presence of E. multilocularis infection in domestic dogs in Edmonton although further work is required to fully understand the risk factors that may contribute to infection and potential transmission to humans.

Prevalence of Fox Tapeworm in Invasive Muskrats in Flanders (North Belgium).

Simple SummaryFox tapeworms are pathogens that need two hosts to complete their life cycle. The reproductive part of the cycle takes place in foxes and the intermediate step usually occurs in rodents. Muskrats can also act as the intermediate host for fox tapeworms. This increases the total number of potential hosts which can also increase the number of pathogens in the environment. In Belgium, where muskrats are non-native, this is one of the ways in which an invasive alien species can have a negative effect on its new environment. From 2009 to 2017 all muskrats caught in Flanders and across the border with Wallonia and France were collected and dissected to estimate how often they were infected with fox tapeworms. Visual examination of the livers of 15,402 muskrats revealed 202 infected animals (1.31%). In Flanders, we found 82 infected animals out of 9421 (0.87%). The percentage of infected animals did not increase during the research period. All the infected animals in Flanders were found in municipalities along the Walloon border. We did not observe a northward spread of EM infection from Wallonia to Flanders.One way in which invasive alien species affect their environment is by acting as pathogen hosts. Pathogens limited by the availability of the native host species can profit from the presence of additional hosts. The muskrat (Ondatra zibethicus) is known to act as an intermediate host for the fox tapeworm (Echinococcus multilocularis). From 2009 to 2017, 15,402 muskrats caught in Flanders and across the border with Wallonia and France were collected and dissected with the aim of understanding the prevalence of this parasite in muskrats. Visual examination of the livers revealed 202 infected animals (1.31%). Out of the 9421 animals caught in Flanders, we found 82 individuals (0.87%) infected with E. multilocularis. No increase in prevalence was observed during this study. All of the infected animals in Flanders were found in municipalities along the Walloon border. We did not observe a northward spread of E. multilocularis infection from Wallonia to Flanders. We hypothesise that the low prevalence is the result of the reduced availability of intermediate hosts and the successful control programme which is keeping muskrat densities in the centre of the region at low levels and is preventing influx from other areas. Our results illustrate that muskrats are good sentinels for E. multilocularis and regular screening can gain valuable insight into the spread of this zoonosis.

CircRNA Expression Pattern and circRNA-miRNA-mRNA Network in HCs, HSCs, and KCs of Murine Liver After Echinococcus multilocularis Infection.

Caused by Echinococcus multilocularis (E. multilocularis), alveolar echinococcosis is reported every year around the world and severely threatens the safety of human beings and animals. However, the molecular interaction relationships between host and E. multilocularis still remains unclear. With multiple functions, circRNA plays a crucial role in regulating the development of a parasitic disease. With that in mind, the main purpose of this study was to reveal the circRNA expression profiles and circRNA–miRNA–mRNA network relationships in hepatocytes (HCs), hepatic stellate cells (HSCs), and Kupffer cells (KCs) of murine liver after E. multilocularis infection. After sequencing, 6,290 circRNAs were identified from 12 hepatic cell samples. Based on the subsequent analysis, 426 and 372 circRNAs were significantly different in HC expression at 2 and 3 months after E. multilocularis infection, and similar results were also demonstrated in HSCs (426 and 372 circRNAs) and KCs (429 and 331 circRNAs), respectively. Eight candidate circRNAs were randomly selected to identify the accuracy of the sequencing results by using qRT-PCR. Additionally, three circRNAs–miRNA–mRNA networks in HCs, HSCs, and KCs were constructed. Taken together, our study provided a systematic presentation of circRNAs in murine liver cells after E. multilocularis infection, and these networks are essential for research in circRNAs associated with E. multilocularis infection.

Integrative Analysis of RNA Expression and Regulatory Networks in Mice Liver Infected by Echinococcus multilocularis.

The larvae of Echinococcus multilocularis causes alveolar echinococcosis, which poses a great threat to the public health. However, the molecular mechanisms underlying the host and parasite interactions are still unclear. Exploring the transcriptomic maps of mRNA, miRNA and lncRNA expressed in the liver in response to E. multilocularis infection will help us to understand its pathogenesis. Using liver perfusion, different cell populations including the hepatic cells, hepatic stellate cells and Kupffer cells were isolated from mice interperitoneally inoculated with protoscoleces. Their transcriptional profiles including lncRNAs, miRNAs and mRNAs were done by RNA-seq. Among these cell populations, the most differentially-expressed (DE) mRNA, lncRNAs and miRNAs were annotated and may involve in the pathological processes, mainly including metabolic disorders, immune responses and liver fibrosis. Following the integrative analysis of 38 differentially-expressed DEmiRNAs and 8 DElncRNAs, the lncRNA-mRNA-miRNA networks were constructed, including F63-miR-223-3p-Fbxw7/ZFP36/map1b, F63-miR-27-5p-Tdrd6/Dip2c/Wdfy4 and IFNgAS1-IFN-γ. These results unveil the presence of several potential lncRNA-mRNA-miRNA axes during E. multilocularis infection, and further exploring of these axes may contribute to better understanding of the pathogenic mechanisms.

Echinococcus multilocularis drives the polarization of macrophages by regulating the RhoA-MAPK signaling pathway and thus affects liver fibrosis

ABSTRACT Echinococcus multilocularis is a small parasite that causes alveolar echinococcosis. It primarily induces liver disorder, such as liver fibrosis and even liver cancer, which severely endangers human lives. This study aims to explore the efficacy of Echinococcus multilocularis soluble antigen in preventing and alleviating alveolar echinococcosis-induced liver fibrosis and determine the underlying mechanism. We first identified the optimal dose and time of Echinococcus multilocularis soluble antigen. The protein levels of key genes in the RhoA-MAPK signaling pathway were remarkably upregulated in RAW264.7 and Ana-1 cells induced with 80 μg/mL Echinococcus multilocularis soluble antigen for 8 h. Interestingly, the upregulated expression levels were remarkably reversed by the RhoA, JNK, ERK, or p38 inhibitor, confirming the significance of the RhoA-MAPK signaling pathway. In addition, the relative contents of M2 polarization markers IL-10 and Arg-1 in macrophages induced with 80 μg/mL Echinococcus multilocularis soluble antigen for 8 h increased, whereas those of M1 polarization markers IL-12 and NOS-2 decreased. Mouse hepatic stellate cells were the key components of the hepatocellular carcinoma tumor microenvironment. Hepatic stellate cells were activated by Echinococcus multilocularis soluble antigen and transformed into the morphology of myofibroblasts in response to liver disorders. By detecting the marker of myofibroblast formation, RhoA inhibitor remarkably reduced the positive expression of α-SMA in mouse hepatic stellate cells induced with Echinococcus multilocularis soluble antigen. Therefore, Echinococcus multilocularis soluble antigen remarkably activated the RhoA-MAPK pathways in macrophages, further inducing the polarization of macrophages and ultimately causing liver fibrosis. Hypothesis: We hypothesize that infection with Echinococcus multilocularis activates the RhoA-MAPK signaling pathway and subsequently induces macrophage polarization to promote hepatic stellate cells activation leading to liver fibrosis. Aims: To investigate the mechanism by which soluble antigen of Echinococcus multilocularis affects liver fibrosis through the RhoA-MAPK pathway driving polarization of macrophages. Goals: To identify new pathways of intervention and drug targets for the regulation of macrophage polarity phenotype switching and the attenuation or inhibition of the development and treatment of liver fibrosis caused by Echinococcus multilocularis infection.

Albendazole reduces hepatic inflammation and endoplasmic reticulum-stress in a mouse model of chronic Echinococcus multilocularis infection.

BackgroundEchinococcus multilocularis causes alveolar echinococcosis (AE), a rising zoonotic disease in the northern hemisphere. Treatment of this fatal disease is limited to chemotherapy using benzimidazoles and surgical intervention, with frequent disease recurrence in cases without radical surgery. Elucidating the molecular mechanisms underlying E. multilocularis infections and host-parasite interactions ultimately aids developing novel therapeutic options. This study explored an involvement of unfolded protein response (UPR) and endoplasmic reticulum-stress (ERS) during E. multilocularis infection in mice.MethodsE. multilocularis- and mock-infected C57BL/6 mice were subdivided into vehicle, albendazole (ABZ) and anti-programmed death ligand 1 (αPD-L1) treated groups. To mimic a chronic infection, treatments of mice started six weeks post i.p. infection and continued for another eight weeks. Liver tissue was then collected to examine inflammatory cytokines and the expression of UPR- and ERS-related genes.ResultsE. multilocularis infection led to an upregulation of UPR- and ERS-related proteins in the liver, including ATF6, CHOP, GRP78, ERp72, H6PD and calreticulin, whilst PERK and its target eIF2α were not affected, and IRE1α and ATF4 were downregulated. ABZ treatment in E. multilocularis infected mice reversed, or at least tended to reverse, these protein expression changes to levels seen in mock-infected mice. Furthermore, ABZ treatment reversed the elevated levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the liver of infected mice. Similar to ABZ, αPD-L1 immune-treatment tended to reverse the increased CHOP and decreased ATF4 and IRE1α expression levels.Conclusions and significanceAE caused chronic inflammation, UPR activation and ERS in mice. The E. multilocularis-induced inflammation and consecutive ERS was ameliorated by ABZ and αPD-L1 treatment, indicating their effectiveness to inhibit parasite proliferation and downregulate its activity status. Neither ABZ nor αPD-L1 themselves affected UPR in control mice. Further research is needed to elucidate the link between inflammation, UPR and ERS, and if these pathways offer potential for improved therapies of patients with AE.

Role of Cytokines on the Progression of Liver Fibrosis in Mice Infected with Echinococcus multilocularis.

BackgroundLiver fibrosis is a significant pathological change of Echinococcus multilocularis (E. multilocularis) infection. This study aimed to explore the role of cytokines on the progression of liver fibrosis in mice infected with E. multilocularis.MethodsLiver histopathological features at 2, 8, 30, 90 and 180 d were quantified by inflammatory severity score. The expression levels of inflammatory cytokines, fibrosis-related cytokines and hepatic cell apoptosis were measured using qRT-PCR and immunohistochemistry.ResultsAt the early stage of infection, parasite stimulation triggers the rapid recruitment of immune cells, such as macrophages and neutrophils. These infiltrated immune cells then produce a large number of cytokines, such as iNOS (inducible nitric oxide synthase), a pro-inflammatory cytokine; TGF-β (transforming growth factor) activated HSCs (hepatic stellate cells) to promote the proliferation of fibroblasts and secretion of ECM (extracellular matrix); MMP9 (matrix metalloproteinase 9) degraded basal ECM and facilitated its replacement by a highly dense interstitial matrix. At the middle and late stages of infection, the expression of IL-10 (interleukin-10) with general inhibitory effect was increased. The imbalance of fiber formation and degradation aggravated liver fibrosis. Meanwhile, the whole process of E. multilocularis infection was accompanied by the necrosis and apoptosis of hepatic cells.ConclusionAlong with the expansion of parasitic infection, dynamic changes in cytokine expression were observed on the liver fibrosis progression, which is helpful to provide some new ideas for the prevention and treatment of liver fibrosis in mice infected with E. multilocularis.

Annual assessment of Echinococcus multilocularis surveillance reports submitted in 2021 in the context of Commission Delegated Regulation (EU) 2018/772.

This report is part of the Echinococcus multilocularis surveillance scientific reports which are presented annually by EFSA to the European Commission and are intended to assess the sampling strategy, data collection and detection methods used by Finland, Ireland, United Kingdom (Northern Ireland) and Norway in their respective surveillance programmes. The surveillance programmes of these four countries were evaluated by checking the information submitted by each of them and verifying that the technical requirements were fulfilled as laid down in Commission Delegated Regulation (EU) 2018/772 of 21 November 2017 supplementing Regulation (EU) No 576/2013 of the European Parliament and of the Council with regard to preventive health measures for the control of Echinococcus multilocularis infection in dogs, and repealing Delegated Regulation (EU) No 1152/2011. The information was divided in four different categories for assessment: the type and sensitivity of the detection method, the selection of the target population, the sampling strategy and the methodology. For each category, the main aspects that need to be considered in order to accomplish the technical requirements of the legislation were checked against compliance of several criteria. Three of the countries participating in this surveillance (Finland, Ireland and Norway (mainland)) succeeded in the fulfilment of the technical legal requirements foreseen in Commission Delegated Regulation (EU) 2018/772 concerning these four different categories. Northern Ireland did not fulfil those requirements, not even assuming a diagnostic test sensitivity value of 0.99 (value provided by the national reference laboratory, higher than the conservative sensitivity value suggested by EFSA, i.e. 0.78). None of the four countries recorded positive samples in the 12‐month reporting period.