As a novel anti-tumor protein recently identified from Trichinella spiralis (T. spiralis) via cDNA display library screening, T. spiralis anti-tumor protein (TspATP) exhibits remarkable potential for inducing immunogenic cell death (ICD). To harness this mechanism for cancer therapy, we engineered mesoporous polydopamine (MPDA)@TspATP nanoparticles through a self-assembly strategy, integrating photothermal capability with ICD induction. Systematic evaluation revealed that NIR irradiation-activated MPDA@TspATP significantly suppressed HepG2 hepatoma cell proliferation and migration. Mechanistically, MPDA@TspATP triggered ICD hallmarks, including enhanced calreticulin (CRT) translocation and HMGB1 release, which subsequently promoted dendritic cell maturation. This dual-modality approach not only amplified tumor cell elimination in vivo (tumor inhibition rate: 79.68 % vs. 45.53 % in TspATP-only group) but also induced a systemic immune response. Our findings position MPDA@TspATP as a pioneering ICD-inducing nanoplatform, offering a synergistic strategy to address tumor recurrence and metastatic progression.

As an intracellular parasitic nematode, Trichinella spiralis (T. spiralis) can induce the formation of nurse cells (NC) in host muscles and keep it to survive within the NC for an extended period. The formation of NC is similar to muscle cell injury and repair which lead to the arrest of satellite cells in the G2/M phase and build a suitable parasitic environment for the muscle larvae of T. spiralis. However, the molecular mechanisms involved in skeletal muscle repair through skeletal muscle satellite cells (SMSC) and the host immune response during T. spiralis infection have not been fully elucidated. In this study, histopathological examination revealed that the severity of damage increased as the infection progressed in the soleus muscle. SMSCs were isolated from BALB/c mice infected with T. spiralis at 4, 21 and 35 days post-infection (dpi). The immunological characteristics of these cells were analyzed by real-time PCR and flow cytometry (FCM). FCM analysis revealed a notable increase in the expression of B7 homolog 1 (B7-H1) in SMSCs following T. spiralis infection, while conversely, the expression of inducible costimulatory ligand (ICOSL) significantly decreased. Furthermore, real-time PCR results showed that toll like receptor 3 (TLR3) expression in SMSCs of the infected mice was upregulated at 21 dpi. The expression levels of three subtypes (PPARα, PPARβ and PPARγ) of peroxisome proliferator-activated receptors (PPARs) also increased in the cells. This study highlights the immunological regulation significance of SMSCs host during T. spiralis infection and suggests that SMSCs actively participant in the local immune response to T. spiralis by regulating the interaction between the parasite and the host.

Background Trichinella spiralis can cause animal and human trichinellosis, which is fatal for human beings. Study demonstrated that toll-like receptor 3 (TLR3) agonist was effective in reducing trichinella infections. Hypericin (Hyp) has great potential in activating TLR3 and may be a favorable choice for immunotherapy of trichinellosis. However, its applications are hampered by poor water solubility and dose-dependent phototoxicity. Purpose This study aimed to overcome the disadvantage of Hyp by using thermo-responsive hydrogel, better exert its immunotherapeutic efficacy against T. spiralis by activating TLR3. Study design and methods We used P(NIPAM-co-AM) hydrogel prepared by acrylamide (AM) and N-isopropylacrylamide (NIPAM) to load Hyp, and named P(NIPAM-co-AM)/Hyp. Subsequently, its lower critical solution temperature (LCST) characterization, biocompatibility, immunomodulatory activity via TLR3 signaling pathway, and therapeutic efficacy against T. spiralis were evaluated. Results The study showed that the controllable drug release rate of P(NIPAM-co-AM)/Hyp owed to its remarkable temperature sensitivity. P(NIPAM-co-AM)/Hyp exhibited exceptional efficacy in activating the TLR3 signaling pathway and significantly promoting DC cells maturation. P(NIPAM-co-AM)/Hyp effectively elicited a robust pro-inflammatory immune response with up-regulation of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-12 (IL-12) in T. spiralis-infected mice. Compared to Hyp and albendazole (ABZ), P(NIPAM-co-AM)/Hyp exhibited a significant decrease in the encysted muscle larvae number and histopathological destruction. The muscle larvae burden was dropped from 58733 to 32833 per mice at a dose of 10 mg/kg, with a reduction rate of 42.8%. Moreover, the reduction rate increased to 64.30% at a dose of 20 mg/kg. Conclusions : This study confirms the therapeutic efficacy of P(NIPAM-co-AM)/Hyp as a TLR3 agonist and provides a new study direction for immunotherapy strategy and vaccine development by targeting parasites.

Trichinella spiralis (T. spiralis) has been reported to induce inflammation, which can cause immune system dysregulation. Myeloid differentiation primary response gene 88 (MyD88) is implicated in inflammation signalling pathways. TJ-M2010-5 is a novel MyD88 inhibitor with remarkable protective effects against several diseases. However, the precise mechanism of TJ-M2010-5’s involvement in spleen impairment and inflammation in the early infection of T. spiralis has yet to be fully elucidated. This study analysed histological, inflammation, and macrophage polarisation of the early T. spiralis-infected mice treated with TJ-M2010-5. MyD88 promoter methylation results showed that the methylation levels in the 5 d group were lower compared to the control group (P < 0.05). Furthermore, the methylation led to an imbalance in anti-inflammatory regulation in the infected mice. After TJ-M2010-5 treatment, spleen impairment was reduced. Sequencing analysis showed that TJ-M2010-5 significantly up-regulated 9 and down-regulated 10 miRNAs compared with the 5 d group. A dual-luciferase reporter assay further revealed that miR-136-5p is involved in the TJ-M2010-5 treatment by targeting AKT3. In RAW264.7 cells, TJ-M2010-5 pre-treatment significantly reversed the M1 polarisation and inhibited nitric oxide (NO) production. LC–MS/MS results showed TJ-M2010-5 was hepatosplenic-targeted. In conclusion, the study demonstrates that TJ-M2010-5 could effectively alleviate spleen impairment and reduce inflammation in mice infected with T. spiralis in its early stages by blocking the activation of PI3K/miR-136-5p/AKT3.

Metacestodiasis is an infectious disease caused by the larval stage of cestode parasites. This disease poses a serious health hazard to wildlife, livestock, and humans, and it incurs substantial economic losses by impacting the safety of the livestock industry, the quality of meat production, and public health security. Unfortunately, there is currently no available molecular diagnostic method capable of distinguishing cysticercus- and Echinococcus-derived microRNAs (miRNAs) from other helminthes and hosts in the plasma of metacestode-infected animals. This study aims to develop a specific, sensitive, and cost-efficient molecular diagnostic method for cysticercosis and echinococcosis, particularly for early detection. The study developed a rolling circular amplification (RCA)-assisted CRISPR/Cas9 detection method based on parasite-derived miRNA let-7-5p. Using a series of dilutions of the let-7 standard, the limit of detection (LOD) of the qPCR, RCA, and RCA-assisted CRISPR/Cas9 methods was compared. The specificity of qPCR and CRISPR/Cas9 was evaluated using four artificially synthesized let-7 standards from different species. A total of 151 plasma samples were used to evaluate the diagnostic performance. Additionally, the study also assessed the correlation between plasma levels of let-7-5p, the number of Taenia pisiformis cysticerci, and the weight of Echinococcus multilocularis cysts. The results demonstrated that the RCA-assisted CRISPR/Cas9 assay could significantly distinguish let-7 from cestodes and other species, achieving a LOD of 10 aM; the diagnostic sensitivity and specificity for rabbit cysticercosis and mouse E. multilocularis were 100% and 97.67%, and 100% and 100%, respectively. Notably, let-7-5p gradually increased in the plasma of T. pisiformis-infected rabbits from 15 days post infection (dpi), peaked at 60 dpi, and persisted until 120 dpi. In E. multilocularis-infected mice, let-7-5p gradually increased from 15 dpi and persisted until 90 dpi. Furthermore, the expression of let-7-5p positively correlated with the number of cysticerci and cyst weight. These results indicated that the let-7-5p-based RCA-assisted CRISPR/Cas9 assay is a sensitive and specific detection method that can be used as a universal diagnostic method for metacestodiasis, particularly for early diagnosis (15 dpi).

Alveolar echinococcosis (AE) is an important infectious disease caused by the metacestode larvae of Echinococcus multilocularis, seriously threatening global public health security. Kupffer cells (KCs) play important roles in liver inflammatory response. However, their role in hepatic alveolar echinococcosis has not yet been fully elucidated. In this study, qRT-PCR was used to detect the expression level of miR-374b-5p in KCs. The target gene of miR-374b-5p was identified through luciferase reporter assays and loss of function and gains. Critical genes involved in NFκB signaling pathway were analyzed by qRT-PCR and western blot. This study reported that miR-374b-5p was significantly upregulated in KCs during E. multilocularis infection and further showed that miR-374b-5p was able to bind to the 3'-UTR of the C/EBP β gene and suppressed its expression. The expression levels of NF-κBp65, p-NF-κBp65 and pro-inflammatory factors including iNOS, TNFα and IL6 were attenuated after overexpression of miR-374b-5p while enhanced after suppression of miR-374b-5p. However, the Arg1 expression level was promoted after overexpression of miR-374b-5p while suppressed after downregulation of miR-374b-5p. Additionally, increased protein levels of NF-κBp65 and p-NF-κBp65 were found in the C/EBP β-overexpressed KCs. These results demonstrated that miR-374b-5p probably regulated the expression of inflammatory factors via C/EBP β/NF-κB signaling. This finding is helpful to explore the mechanism of inflammation regulation during E. multilocularis infection.

Cysticercosis pisiformis, a highly prevalent parasitic disease worldwide, causes significant economic losses in the rabbit breeding industry. Previous investigations have identified a novel microRNA, designated as novel-miR1, within the serum of rabbit infected with Cysticercus pisiformis. In the present study, we found that C. pisiformis-derived novel-miR1 was released into the rabbit serum via exosomes. Through computational analysis using TargetScan, miRanda, and PITA, a total of 634 target genes of novel-miR1 were predicted. To elucidate the functional role of novel-miR1, a dual-luciferase reporter assay was utilized and demonstrated that novel-miR1 targets rabbit Toll-like receptor 2 (TLR2). Rabbit peripheral blood lymphocytes (PBLCs) were transfected with novel-miR1 mimic and mimic NC, and the in vitro experiments confirmed that novel-miR1 suppressed the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 through the nuclear factor kappa B (NF-κB) pathway. In vivo experiments demonstrated that novel-miR1 was significantly upregulated during the 1–3 months following infection with C. pisiformis in rabbits. Notably, this upregulation coincided with a downregulation of TLR2, P65, pP65, TNF-α, IL-1β, and IL-6 in PBLCs. Collectively, these results indicate that the novel-miR1 derived from C. pisiformis inhibited the rabbits’ immune response by suppressing the NF-κB-mediated immune response. This immune modulation facilitates parasite invasion, survival, and establishment of a persistent infection.

旨在探讨多房棘球蚴感染早期对小鼠肝脂质代谢的影响.选取60只清洁级BALB/c小鼠(6周龄,雄性)随机分为试验组和对照组,每组30只.试验组小鼠每只腹腔接种600个原头蚴,对照组小鼠腹腔注射等体积的PBS缓冲液.采用原位两步循环灌流及多步低速差速离心分离纯化肝实质细胞.利用高通量测序分析感染3月小鼠肝实质细胞差异表达的mRNA,并对其进行KEGG、GO分析和qRT-PCR验证.制备多房棘球蚴感染2、3和6月小鼠的肝组织切片,分别进行油红O染色,并通过qRT-PCR检测多房棘球蚴感染不同感染时期肝实质细胞脂质代谢相关基因的表达水平.结果表明,与未感染对照组相比,小鼠感染多房棘球蚴后3、6月肝脂肪沉积明显增多;感染2月的肝实质细胞脂质合成相关基因Scd1、Fasn和Srebf1,以及脂质氧化分解相关基因Acox1和Cpt1α均明显上调;感染3月的肝实质细胞Scd1、Fasn、Srebf1及Cd36的表达水平均明显升高,而Acox1、Cpt1α、Pparγ和Pparα的表达水平明显降低.上述结果表明,多房棘球蚴感染早期可能通过促进肝实质细胞的脂质合成,抑制脂质氧化分解,从而导致肝脂质沉积.

Infection of Taenia pisiformis cysticercus is very frequently found in lagomorphs and causes serious economic losses to rabbit breeding industry. T. pisiformis cysticercus has evolved numerous strategies to manipulate their hosts. The release of exosomes is of importance in the interaction between host and parasite. However, the mechanism by which T. pisiformis cysticercus evades the host immune system for long-term survival within the host remains unclear. Using small RNA sequencing and TMT labelling proteomic, we profiled the expression patterns of miRNAs and proteins in rabbit peritoneal macrophages treated with T. pisiformis cysticercus exosomes. Seven differentially expressed (DE)-miRNAs and six DE-proteins were randomly selected to validate the accuracy of the sequencing data by qRT-PCR or western blot. Functions of DE-miRNAs and proteins were analyzed using public data bases. And DE-miRNAs-DE-proteins correlation network were established. CCK-8 assay was used to evaluate the effect of exosomes on macrophages proliferation. Cell cycle of macrophages, isolated from T. pisiformis-infected rabbits, was determined using flow cytometry. A total of 21 miRNAs were significantly differentially expressed, including three worm-derived miRNAs. The expressions of miRNAs and proteins were consistent with the sequencing results. DE-miRNAs targets were related to cell proliferation and apoptosis. Exosomes treatment resulted in a decrease of macrophages proliferation. In vivo, T. pisiformis cysticercus significantly induced S phase cell arrest. Moreover, DE-proteins were related to production of interferon-gamma and interleukin-12, and immunoregulation. Correlation network analysis revealed a negative correlation relationship between DE-miRNAs and DE-proteins. Among them, novel334 and tpi-let-7-5p have potential regulatory effects on IL1β and NFκB2 respectively, which imply that novel334-IL1β/tpi-let-7-5p-NFκB2 axis may be an important way that T. pisiformis cysticercus modulates host immune response through exosomes. Further understanding of these potential regulatory mechanisms will contribute to clarify the mechanism of escape mediated by T. pisiformis exosomes.

目的 建立检测家兔豆状囊尾蚴感染的滚环扩增(RCA)方法.方法 以家兔血清中豆状囊尾蚴来源的novel-miR1为诊断靶标,设计连接序列和锁式探针,并对连接序列和锁式探针的比例、反应时间、酶用量、dNTP用量以及扩增反应时间等5个重要条件进行优化,建立检测豆状囊尾蚴感染的RCA方法.将novel-miR1标准品倍比稀释为1 fmol/L至100 nmol/L的9个不同浓度的样品,评价RCA方法的灵敏度.用优化后的RCA方法分别检测健康家兔和豆状囊尾蚴感染家兔血清miRNA各20份(实验室保存样品),并通过受试者工作特征(ROC)曲线分析其敏感性和特异性.20只雌性家兔每只经口感染1 000个豆状带绦虫虫卵,采集感染前和感染后每个月的家兔血样制备血清miRNA,用优化后的RCA方法进行检测,评价其应用效果.结果 电泳结果显示,RCA扩增产物的DNA停留在加样孔中,形成一条明亮条带.反应条件优化试验结果显示,连接序列浓度为2μmol/L、锁式探针浓度为1μmol/L(连接序列和锁式探针的比为2:1)、T4 DNA连接酶为350 U、连接180 min时连接效率最高.在100μl的扩增体系中,dNTP浓度为0.5 μmol/L,扩增240 min时,RCA扩增效果最佳.优化后RCA的最低检测浓度为10 pmoI/L.RCA检测健康家兔和豆状囊尾蚴感染家兔血清miRNA样品的平均荧光值分别为53.298±1.707和97.498±5.892,差异有统计学意义(t=7.206,P＜0.01).ROC分析结果显示,当阳性临界值为61.69时,RCA方法的敏感性和特异性均为95％,AUC为0.955 0,似然比为19.00.RCA检测豆状囊尾蚴感染后不同时间的家兔血清miRNA结果显示,20份感染前的血清中19份检测结果为阴性,1份为阳性;感染后1个月的血清中,17份为阳性,2份为疑似,1份为阴性;感染后2个月的血清,1份为阴性,其余为阳性;感染后3个月的血清,3份为阴性,其余为阳性.结论 建立了检测家兔豆状囊尾蚴感染的RCA方法,该方法在豆状囊尾蚴感染后3个月内的家兔血清中检出novel-miR1,具有良好的应用潜力..

目的 建立豆状囊尾蚴体外长期培养方法,并对其培养分泌产物进行鉴定.方法 将豆状带绦虫虫卵经口感染新西兰白兔(2 000枚/只),感染后3个月,从感染兔腹腔中收集豆状囊尾蚴.将虫体随机分为PBS组、RPMI1640组、胆汁组(RPMI1640+10％兔胆汁)和血清组(RPMI1640+10％无外泌体胎牛血清),10个/组,37℃、5％CO2培养后5min、1h、2h、12h、24 h、36 h、48 h及其后每天进行观察,记录豆状囊尾蚴头节翻出情况、虫体活力、体外存活时间及形态改变等,筛选适宜的培养条件.取50个幼虫在细胞瓶(25 cm2)中按筛选出的培养条件培养,每隔48小时收集培养液离心浓缩,取上清,蛋白质免疫印迹(Western blotting)检测豆状带绦虫烯醇化酶(Tpeno)和Tp14-3-3蛋白的表达情况.收集豆状囊尾蚴培养上清,提取外泌体,分别与阴性兔血清、CD63抗体、抗Tpeno单克隆抗体1D7和抗Tp14-3-3多克隆抗体孵育45 min,再加入胶体金标记的IgG,透射电镜观察外泌体形态,免疫电镜检测其标记蛋白CD63、Tpeno和Tp14-3-3表达情况.采用SPSS 20.0统计学软件进行数据分析,组间比较采用单因素方差分析.结果 培养前的豆状囊尾蚴呈椭圆形,有内凹的点状乳白色头节;PBS组可存活7d;RPMI 1640组可存活2个月;胆汁组存活时间最短,仅48h;血清组幼虫存活时间最长,可达3个月以上,高于其他3个组(P＜0.05).存活期内,PBS组、RPMI 1640组、胆汁组和血清组幼虫囊泡长度分别为(1.38±0.39)、(1.30±0.12)、(1.18±0.59)和(1.83±0.10)cm;宽度分别为(0.45±0.10)、(0.68±0.05)、(0.25±0.06)和(0.85±0.05)cm;血清组虫体囊泡长度和宽度与其余3组相比差异均有统计学意义(F长 度=7.58、65.93、7.11,F宽度=73.85、29.41、308.57,P＜0.05 或P＜0.01).培养 12 h 时,PBS组、RPMI 1640组、胆汁组和血清组幼虫头节翻出率分别为(33.3±5.8)％、(20.0±0.0)％、(100.0±0.0)％、(13.3±5.8)％,其中血清组与PBS和胆汁组头节翻出率差异有统计学意义(F=18.00、676.00,P＜0.01).对各组培养效果比较显示,血清组的培养液(RPMI 1640+10％无外泌体胎牛血清)为最适培养液.SDS-PAGE电泳分析结果显示,利用RPMI 1640+10％无外泌体胎牛血清培养豆状囊尾蚴,可成功提取到虫体的代谢_分泌产物和外泌体蛋白.Western blotting分析结果显示,用特异性抗体检测可见Tpeno和Tp14-3-3阳性条带,相对分子质量(Mr)分别约47 000和28 000,与预期相符.透射电镜下豆状囊尾蚴外泌体大小不一,直径为50～150 nm,为圆形或椭圆形的囊泡.免疫电镜分析显示,阴性兔血清不能识别外泌体蛋白,未见胶体金颗粒标记;外泌体标志蛋白CD63、虫体特异性蛋白Tpeno和Tp14-3-3均为阳性表达,胶体金颗粒标记明显.结论 初步建立了豆状囊尾蚴体外培养体系,可连续培养豆状囊尾蚴至少3个月以上.采用该体外培养方法培养可在培养上清中成功提取豆状囊尾蚴的外泌体.

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.

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.

多房棘球蚴(Em)是一种危害严重的人畜共患寄生虫.为探索非编码RNA在多房棘球蚴感染诱导小鼠肝枯否氏细胞(KCs)极化中的作用机制,本研究用多房棘球蚴原头蚴感染BALB/c小鼠,分离感染2月小鼠肝KCs,通过RNA测序分析,构建与多房棘球蚴感染引起的KCs M2型极化相关的竞争性内源RNA(competing endogenous RNAs,ceRNA)调控网络,并通过qPCR和miRNA过表达进行验证.结果表明,多房棘球蚴感染小鼠2月后,KCs M1型标志分子均显著下调,而M2型标志分子均显著上调.5个circRNA和9个miRNA参与M2型标志分子IL10、IL13、Arg1基因的表达调控,而且其表达趋势与测序结果及circRNA-miRNA-mRNA调节关系基本一致.另外,miR-466c-5p过表达导致了 circ_0000372和IL13的表达下调,说明可能存在circ_0000372-miR-466c-5p-IL13轴的调节关系,并在多房棘球蚴感染诱导小鼠肝KCs极化中发挥重要作用.本研究为进一步揭示多房棘球蚴调控宿主巨噬细胞极化的相关机制提供了重要理论依据.

Additional file 4: Table S5. KEGG interaction network analysis of differentially expressed mRNAs.

Cysticercus pisiformis, the larval stage of Taenia pisiformis, causes serious illness in rabbits that severely impacts the rabbit breeding industry. An inhibitive Th2 immune response can be induced by let-7-enriched exosomes derived from T. pisiformis cysticercus. However, the underlying molecular mechanisms are not completely understood. Here, we report that exosomal miR-let-7-5p released by T. pisiformis cysticercus played a critical role in the activation of M2 macrophages. We found that overexpression of let-7-5p in M1 macrophages decreased M1 phenotype expression while promoting polarization to the M2 phenotype, which is consistent with experimental data in exosome-treated macrophages alone. In contrast, knockdown of let-7-5p in exosome-like vesicles promoted M1 polarization and decreased M2 phenotype expression. Furthermore, down-regulation of transcription factor CCAAT/enhancer-binding protein (C/EBP)-δ resulted in the decrease of M1 phenotype markers and increase of M2 phenotype markers. These results suggested that let-7 enriched in exosome-like vesicles from T. pisiformis metacestodes can induce M2 macrophage polarization via targeting C/EBP δ, which may be involved in macrophage polarization induced by T. pisiformis metacestodes. The finding helps to expand our knowledge of the molecular mechanism of immunosuppression and Th2 immune response induced by metacestodes.

BACKGROUND:Bovine viral diarrhea virus (BVDV) is a major pathogen that causes bovine viral diarrhea/mucosal disease (BVD-MD), which has become a global infectious disease due to its wide spread and the lack of effective treatment. The process of BVDV infection is complex. Once infected, host immune cells are activated and modulated. As a major immune cell, peripheral blood lymphocyte cells (PBLCs) are the primary target of BVDV. In order to further understand the mechanism of BVDV- host interaction, the expression profiles of host lymphocytes mRNAs associated with BVDV infection were investigated by transcriptomic sequencing analysis. RESULTS:The transcriptomic sequencing analysis was performed on bovine PBLCs infected with CP BVDV-2 GS2018 after 12 h of infection. Gene expression profiling demonstrated that 1052 genes were differentially expressed in GS2018 infected PBLCs compared with the control group. Of these genes, 485 genes were up-regulated and 567 were down-regulated. The 19 differential expressed genes (DEGs) were selected for validation using quantitative real-time PCR and the results were consistent with the results of RNA-Seq. Gene ontology enrichment and KEGG pathway analysis showed that 1052 DEGs were significantly enriched in 16 pathways, including cytokine-cytokine receptor interaction, IL17, PI3K-Akt, MAPK and TNF signaling pathway. PPI network analysis showed that IL17A, IFN-γ and TNF-α interacted with various proteins and may play crucial roles in BVDV-2 infection. Of note, we confirmed that GS2018 induced Th17 cell differentiation in PBLCs and persistently increased the expression levels of IL17A. In turn, the replication of GS2018 was inhibited by IL17A. CONCLUSION:In this study, the transcription changes of DEGs related to host immune responses in bovine PBLCs were caused by CP BVDV-2 infection. In particular, the effector molecules IL17A of Th17 cells were significantly up-regulated, which inhibited viral replication. These results will contribute to exploration and further understanding of the host immune response mechanism and interaction between host and BVDV-2.

目的 建立一种多重聚合酶链式反应(multiplex polymerase chain reaction,mPCR)方法,用于快速鉴别猪囊尾蚴等4种猪的绦虫蚴.方法 比较猪带绦虫(Taenia solium)、亚洲带绦虫(Taenia asiatica)、细粒棘球绦虫(Echinococcusgranulosus)和泡状带绦虫(Taenia hydatigena)的线粒体基因组,选取tRNA-Val、tRNA-Ala、tRNA-Asp和nad1基因作为分子检测靶标,设计4条种特异性上游引物(TaF、EgF、ThF、和TsF)和1条下游通用引物(TR),通过优化反应条件和体系,建立了在一个PCR体系中即可完成对4种猪绦虫蚴的快速鉴别的mPCR方法.结果 4种猪绦虫蚴的特异性PCR引物等比例混合后,在退火温度为50℃的条件下,该mPCR方法可同时扩增出C.cellulosae、T.asiatica、E.granulosus和C.tenuicol-lis的特异性基因片段,其大小分别为478 bp、710 bp、619 bp和542 bp.PCR产物测序后经BLAST比对,与同种虫体的基因序列相似性在99％以上.结论 本研究建立的mPCR方法具有很高的种间特异性,并且可通过扩增片段的大小对4种猪绦虫蚴进行快速、准确的鉴别.

Cysticercus pisiformis (C. pisiformis), the larval form of Taenia pisiformis, parasitize mainly the liver, omentum and mesentery of rabbits and cause huge economic losses in the rabbit breeding industry. MicroRNA (miRNA), a short non-coding RNA, is widely and stably distributed in the plasma and serum. Numerous data demonstrates that, after parasitic infection, miRNAs become the key regulatory factor for controlling host biological processes. However, the roles of serum miRNAs in C. pisiformis-infected rabbits have not been elucidated. In this study, we compared miRNA expression profiles between the C. pisiformis-infected and healthy rabbit serum using RNA-seq. A total of 192 miRNAs were differentially expressed (fold change ≥ 2 and p < 0.05), including 79 up- and 113 downregulated miRNAs. These data were verified by qRT-PCR (real time quantitative polymerase chain reaction) analysis. Additionally, GO analysis showed that the target genes of these dysregulated miRNAs were most enriched in cellular, single-organism and metabolic processes. KEGG pathway analysis showed that these miRNAs target genes were involved in PI3K-Akt, viral carcinogenesis and B cell receptor signaling pathways. Interestingly, after aligning clean reads to the T. pisiformis genome, four (miR-124-3p_3, miR-124-3p_4, miR-124a and novel-miR1) T. pisiformis-derived miRNAs were found. Of these, novel-miR1was upregulated in different periods after C. pisiformis infection, which was verified qRT-PCR, and pre- novel-miR-1 was amplified from the cysticerci by RT-PCR, implying novel-miR-1 was derived from C. pisiformis and has great potential for the diagnosis of Cysticercosis pisiformis infection. This is the first investigation of miRNA expression profile and function in the serum of rabbits infected by C. pisiformis, providing fundamental data for developing diagnostic targets for Cysticercosis pisiformis.

Taenia asiatica is a zoonotic parasite found in the human intestine and pig liver that evolved various strategies to survive the host's defenses. Exosomes are membranous vesicles released by cells and are an important vehicle in parasite-host interactions. However, no literature exists on the specific infection mechanisms of T. asiatica against the host defense response, and further research is required to understand the parasite-host interaction. In this study, we investigated the host's differentially expressed genes (DEGs) while stimulating them with exosomes derived from the T. asiatica adult worm (Tas-exo) on LoVo by RNA-seq analysis. Our results identified 348 genes as being significantly differentially expressed for the Tas-exo group when comparing with that of the NC group. Some of these genes are related to modulation of cell proliferation and cell autophagy. Surprisingly, autophagy and cell proliferation have crucial roles in the defense against parasites; accordingly, we detected cell proliferation and autophagy in LoVo cells by CCK8, immunofluorescence, and Western blotting, demonstrating that Tas-exo could inhibit LoVo cell proliferation and autophagy via AMPK pathway. When P62 and p-mTOR/mTOR expression were significantly increased, BeclinI and pAMPK/AMPK were significantly decreased. These results expand our understanding of parasite-host interactions mediated by exosomes.