Induction of hepatic fibrosis in mice with schistosomiasis by extracellular microRNA-30 derived from Schistosoma japonicum eggs.

Background: Schistosomiasis is a zoonotic parasitic disorder induced by the infestation of schistosomes, a genus of trematodes. MicroRNAs (miRNAs) in egg-derived exosomes are crucial for modulating the host's immune responses and orchestrating the pathophysiological mechanisms. Although the exosomes secreted by S. japonicum contain abundant miRNAs, the specific roles of these miRNAs in the pathogenesis of schistosomiasis-induced hepatic fibrosis are yet to be comprehensively elucidated. The egg exosomes of S. japonicum secrete miRNA-30, a novel miRNA. Methods: In vitro, the effect of miRNA-30 was evaluated by transfecting HSCs with miRNA mimics. The target gene biosignature for miRNA-30 was predicted using the miRDB software. The effect of miRNA-30 in hepatic fibrosis was evaluated by either elevating its expression in healthy mice or by inhibiting its activity in infected mice by administration of recombinant adeno-associated virus serotype eight vectors expressing miRNA-30 or miRNA sponges. Results: This novel miRNA can activate hepatic stellate cells (HSCs), the prinary effector cells of hepatic fibrosis, in vitro, i.e., it significantly increases the fibrogenic factors Col1(α1), Col3(α1), and α-SMA at both mRNA and protein levels. In addition, miRNA-30 may activate HSCs by targeting the host RORA gene. In addition, in vivo experiments were conducted by administering a recombinant adeno-associated viral vector to modulate the expression levels of miRNA-30. The overexpression of miRNA-30 in healthy mice significantly elevated the expression of Col1(α1), Col3(α1), and α-SMA at both the transcriptomic and proteomic scales. This overexpression was coupled with a pronounced augmentation in the hepatic hydroxyproline content. Conversely, the in vivo silencing of miRNA-30 in infected mice induced a considerable reduction in the size of hepatic granulomas and areas of collagen deposition. Hence, in vivo, modulation of miRNA-30 expression may play a pivotal role in ameliorating the severity of hepatic fibrosis in mice afflicted with S. japonica. Conclusions: The study results suggest that miRNA-30 may augment schistosomiasis-induced hepatic fibrosis through a probable interaction with the host RORA. Our study may improve the current theoretical framework regarding cross-species regulation by miRNAs of hepatic fibrosis in schistosomiasis.

Molecular identification and genetic characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in human immunodeficiency virus/acquired immunodeficiency syndrome patients in Shanghai, China.

BACKGROUND: Opportunistic infections are a ubiquitous complication in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients. Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans. In China, despite the number of HIV/AIDS patients being extremely large, only a few studies have investigated opportunistic infections caused by intestinal pathogens in this patient population. The aims of this study were to elucidate the occurrence and genetic characteristics of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients. METHODS: We collected fecal specimens from 155 HIV/AIDS patients (one from each patient). All of the specimens were examined for the presence of the pathogens by genotyping using polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene for Cryptosporidium spp.; the triosephosphate isomerase, ß-giardin and glutamate dehydrogenase genes for G. duodenalis; and the internal transcribed spacer region of the rRNA gene for E. bieneusi. The Cryptosporidium-positive specimens were further subtyped by polymerase chain reacion and sequencing of the 60-kDa glycoprotein gene. RESULTS: Six (3.9%), three (1.9%), and eight (5.2%) HIV/AIDS patients were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. No statistical differences were observed in occurrence rate between the groups by gender, clinical symptom (diarrhea), and CD4+ cell count. Four Cryptosporidium species were identified: Cryptosporidium hominis (n = 2), Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). Furthermore, two C. hominis subtypes (IeA12G3T3 and IaA28R4) were detected. Three G. duodenalis-positive specimens were successfully amplified and sequenced at the triosephosphate isomerase and ß-giardin loci, which led to the identification of assemblages C and B, respectively. Seven genotypes (D, Type IV, EbpC, Peru11, EbpD, A, and I) were identified in E. bieneusi-positive specimens. CONCLUSIONS: Our findings should increase awareness of AIDS-related opportunistic intestinal pathogens, and indicate the need for routine examination in clinical practice for the detection of Cryptosporidium spp., G. duodenalis, and E. bieneusi. Homology analyses of the three intestinal pathogens at the nucleotide and/or amino acid levels indicated their zoonotic potential.

The single-cell landscape of cystic echinococcosis in different stages provided insights into endothelial and immune cell heterogeneity.

Introduction: Hydatid cysts and angiogenesis are the key characteristics of cystic echinococcosis, with immune cells and endothelial cells mediating essential roles in disease progression. Recent single-cell analysis studies demonstrated immune cell infiltration after Echinococcus granulosus infection, highlighting the diagnostic and therapeutic potential of targeting certain cell types in the lesion microenvironment. However, more detailed immune mechanisms during different periods of E. granulosus infection were not elucidated. Methods: Herein, we characterized immune and endothelial cells from the liver samples of mice in different stages by single-cell RNA sequencing. Results: We profiled the transcriptomes of 45,199 cells from the liver samples of mice at 1, 3, and 6 months after infection (two replicates) and uninfected wild-type mice. The cells were categorized into 26 clusters with four distinct cell types: natural killer (NK)/T cells, B cells, myeloid cells, and endothelial cells. An SPP1+ macrophage subset with immunosuppressive and pro-angiogenic functions was identified in the late infection stage. Single-cell regulatory network inference and clustering (SCENIC) analysis suggested that Cebpe, Runx3, and Rora were the key regulators of the SPP1+ macrophages. Cell communication analysis revealed that the SPP1+ macrophages interacted with endothelial cells and had pro-angiogenic functions. There was an obvious communicative relationship between SPP1+ macrophages and endothelial cells via Vegfa-Vegfr1/Vegfr2, and SPP1+ macrophages interacted with other immune cells via specific ligand-receptor pairs, which might have contributed to their immunosuppressive function. Discussion: Our comprehensive exploration of the cystic echinococcosis ecosystem and the first discovery of SPP1+ macrophages with infection period specificity provide deeper insights into angiogenesis and the immune evasion mechanisms associated with later stages of infection.

Proteomic profiling of serum extracellular vesicles identifies diagnostic markers for echinococcosis.

Echinococcosis is a parasitic disease caused by the metacestodes of Echinococcus spp. The disease has a long latent period and is largely underdiagnosed, partially because of the lack of effective early diagnostic approaches. Using liquid chromatography-mass spectrometry, we profiled the serum-derived extracellular vesicles (EVs) of E. multilocularis-infected mice and identified three parasite-origin proteins, thioredoxin peroxidase 1 (TPx-1), transitional endoplasmic reticulum ATPase (TER ATPase), and 14-3-3, being continuously released by the parasites into the sera during the infection via EVs. Using ELISA, both TPx-1 and TER ATPase were shown to have a good performance in diagnosis of experimental murine echinococcosis as early as 10 days post infection and of human echinococcosis compared with that of control. Moreover, TER ATPase and TPx-1 were further demonstrated to be suitable for evaluation of the prognosis of patients with treatment. The present study discovers the potential of TER ATPase and TPx-1 as promising diagnostic candidates for echinococcosis.

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.

Genetic Diversity and Haplotype Analysis of Cattle Hydatid Cyst Isolates Using Mitochondrial Markers in Turkey.

Echinococcus granulosus sensu lato (s.l.) causes cystic echinococcosis in ungulates and humans. The current study was designed to find the genetic diversity and haplotypic profiles of hydatid cysts from the lungs of cattle in three provinces in eastern Turkey. Individual cyst isolates (n = 60) were collected from infected cattle lungs after slaughter and then samples were stored in ethanol (70%) until further use. From each isolate, total gDNA was extracted from the cysts' germinal layers. A partial (875 bp) mt-CO1 gene was amplified by PCR and sequenced unidirectionally. The final size of the trimmed sequences was 530 bp for 60 sequences. Sequence and haplotype analyses were performed, followed by phylogenetic analyses. According to BLAST searches, all sequences were detected as E. granulosus s.s. (G1 and G3 strains). Forty-nine point mutations were identified. In addition, five conserved fragments were detected in all sequences. The haplotype analysis diagram showed E. granulosus s.s. haplotypes organized in a star-like configuration. The haplotypes were characterized by 1-17 mutations compared with the fundamental focal haplotype. Thirty-three haplotypes were determined in 60 samples of which 17 (28.3%) belonged to the main haplotype (Hap_06). The mt-CO1 sequences revealed 49 polymorphic sites, 34.5% (20/49) of which were informative according to parsimony analysis.

A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis.

Schistosomiasis caused by Schistosoma japonicum is a serious public health problem in China. Granuloma and hepatic fibrosis are the main pathological features of schistosomiasis japonica. The role and mechanism of egg-derived exosomes of S. japonicum in liver fibrosis remain unclear. In this study, we found that egg-derived exosomes of S. japonicum carry a new type of microRNA (miRNA-33). In vitro, this novel miRNA upregulated the expression of smooth muscle actin (α-SMA) and collagen 1 α1 (Col 1 α1) in the human hepatic stellate cell (LX-2) line at both mRNA and protein levels. In vivo, this novel miRNA was upregulated in the serum of infected mice, and when injected into mice through the tail vein using miRNA agomir, α-SMA, Col 1 α1, and Col 3 α1 were upregulated in liver tissue at both mRNA and protein levels. In addition, this novel miRNA downregulated the expression of α-SMA and Col 1 α1 in liver tissue at mRNA and protein levels in mice infected with S. japonicum and treated with miRNA antagomir. The novel miRNA-33 upregulated TGF-ß Receptor I (TGF-ß RI) at both mRNA and protein levels in LX-2 cells. Our results suggest that this novel miRNA from egg-derived exosomes of S. japonicum can promote liver fibrosis in the host in a cross-species manner, and the degree of fibrosis can be decreased by inhibiting the expression of this miRNA.

cGAS exacerbates Schistosoma japonicum infection in a STING-type I IFN-dependent and independent manner.

Schistosomiasis, which is caused by infection with Schistosoma spp., is characterized by granuloma and fibrosis in response to egg deposition. Pattern recognition receptors are important to sense invading Schistosoma, triggering an innate immune response, and subsequently shaping adaptive immunity. Cyclic GMP-AMP synthase (cGAS) was identified as a major cytosolic DNA sensor, which catalyzes the formation of cyclic GMP-AMP (cGAMP), a critical second messenger for the activation of the adaptor protein stimulator of interferon genes (STING). The engagement of STING by cGAMP leads to the activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and the subsequent type I interferon (IFN) response. cGAS is suggested to regulate infectious diseases, autoimmune diseases, and cancer. However, the function of cGAS in helminth infection is unclear. In this study, we found that Cgas deficiency enhanced the survival of mice infected with S. japonicum markedly, without affecting the egg load in the liver. Consistently, Cgas deletion alleviated liver pathological impairment, reduced egg granuloma formation, and decreased fibrosis severity. In contrast, Sting deletion reduced the formation of egg granulomas markedly, but not liver fibrosis. Notably, Cgas or Sting deficiency reduced the production of IFNß drastically in mice infected with S. japonicum. Intriguingly, intravenous administration of recombinant IFNß exacerbated liver damage and promoted egg granuloma formation, without affecting liver fibrosis. Clodronate liposome-mediated depletion of macrophages indicated that macrophages are the major type of cells contributing to the induction of the type I IFN response during schistosome infection. Moreover, cGAS is important for type I IFN production and phosphorylation of TBK1 and IRF3 in response to stimulation with S. japonicum egg- or adult worm-derived DNA in macrophages. Our results clarified the immunomodulatory effect of cGAS in the regulation of liver granuloma formation during S. japonicum infection, involving sensing schistosome-derived DNA and producing type I IFN. Additionally, we showed that cGAS regulates liver fibrosis in a STING-type I-IFN-independent manner.

Prevalence of Echinococcus Species in Wild Foxes and Stray Dogs in Qinghai Province, China.

Echinococcosis is a zoonotic parasitic disease that is highly endemic to the Qinghai province of China. Limited data are available on the prevalence of the causal pathogen, Echinococcus spp., in definitive hosts in this region. Thus, the aim of this study was to evaluate the prevalence of Echinococcus spp. in wild foxes and stray dogs in Qinghai province. Five hundred and twenty-eight feces from wild foxes and 277 from stray dogs were collected from 11 counties in the Golog, Yushu, and Haixi prefectures and screened for Echinococcus spp. using copro-DNA polymerase chain reaction (PCR). In total, 5.5% of wild foxes and 15.2% of stray dogs tested positive for Echinococcus spp. The prevalence rates of Echinococcus spp. in wild foxes in Golog, Yushu, and Haixi were 7.3%, 5.2%, and 1.9%, respectively. In stray dogs, these rates were 13.3%, 17.3%, and 0%, respectively. Sequencing analysis determined that Echinococcus multilocularis was the most prevalent species, occurring in 4.0% and 12.6% of wild foxes and stray dogs, respectively. Echinococcus shiquicus was observed in 1.5% of wild foxes and 0.7% of stray dogs. Echinococcus granulosus was observed only in wild dogs, with a prevalence rate of 1.8%. To our knowledge, this is the first report on the prevalence of E. shiquicus in dogs in Qinghai province. The current results improve our understanding of the transmission and dissemination of human echinococcosis and suggest that exposure to the eggs of E. multilocularis harbored by wild foxes and stray dogs may pose a great risk of alveolar echinococcosis to humans in Qinghai province.

Prevalence and genetic characteristics of Blastocystis hominis and Cystoisospora belli in HIV/AIDS patients in Guangxi Zhuang Autonomous Region, China.

Blastocystis hominis and Cystoisospora belli are considered to be common opportunistic intestinal protozoa in HIV/AIDS patients. In order to investigate the prevalence and genetic characteristics of B. hominis and C. belli in HIV/AIDS patients, a total of 285 faecal samples were individually collected from HIV/AIDS patients in Guangxi, China. B. hominis and C. belli were investigated by amplifying the barcode region of the SSU rRNA gene and the internal transcribed spacer 1 (ITS-1) region of the rRNA gene, respectively. Chi-square test or Fisher's exact test were conducted to assess the risk factors related to B. hominis and C. belli infection. The prevalence of B. hominis and C. belli was 6.0% (17/285) and 1.1% (3/285) respectively. Four genotypes of B. hominis were detected, with ST3 (n = 8) and ST1 (n = 6) being predominant, followed by ST6 (n = 2) and ST7 (n = 1). Females had a statistically higher prevalence of B. hominis (11.6%) than males (4.2%). The statistical analysis also showed that the prevalence of B. hominis was significantly associated with age group and educational level. Our study provides convincing evidence for the genetic diversity of B. hominis, which indicates its potential zoonotic transmission and is the first report on the molecular characteristics of C. belli in HIV/AIDS patients in China.

Risk Factors for Clonorchis sinensis Infection in Residents of Binyang, Guangxi: A Cross-Sectional and Logistic Analysis Study.

Background: Clonorchiasis is a serious food-borne parasitic disease caused by Clonorchis sinensis infection. C. sinensis, a major fish-borne trematode, is a known causative agent of cholangiocarcinoma. The risk factors for C. sinensis infection include individual eating behaviors and environmental factors. In this study, we evaluated the C. sinensis infection rate and the associated risk factors among residents in Binyang County, Guangxi, China. Methods: In 2016 and 2017, five villages from Binyang, Guangxi were selected by multistage cluster random sampling for a cross-sectional study. A modified Kato-Katz thick smear method was used to examine C. sinensis eggs in fecal samples in triplicate (three smears for each sample). Both uni-variate and multi-variate logistic regression analyses were carried out to identify the risk factors for C. sinensis infection. Results: A total of 1,977 fecal samples were collected from villagers in the investigated areas. The overall infection rates of C. sinensis in Binyang County was 20.49% (405/1,977). The mean age of participants was 39.42 ± 23.48 (range: 3-89 years old), and the highest infection rate (33.72%) was seen in the age group of 40-49 years old, followed by those aged 50-59 (31.83%). Multi-variate logistic regression analysis showed that higher infection rates were significantly associated with males (aOR = 6.51, 95% CI = 4.67-9.08), Zhuang (aOR = 2.41, 95% CI = 1.62-3.59), ages (aOR = 33.51, 95% CI = 10.13-110.86), frequency of raw fresh fish consumption (aOR = 14.56, 95% CI = 9.80-21.63), and close contact with cats and dogs (aOR = 1.53, 95% CI = 1.02-2.30). Occupations and education levels showed no significant association with C. sinensis infection (P > 0.05). Conclusions: High levels of C. sinensis infection were observed among residents in Binyang County, Guangxi. Intervention strategies should be strengthened among the investigated population at high risk, such as males, Zhuang and older individuals, especially those who frequently eat raw fresh fish. In addition, the individuals contacting with cats and/or dogs were observed to have significantly higher infection rate of C. sinensis than those having no contact with cats and dogs. The association between contacting with cats and/or dogs and C. sinensis infection needs to be explored and confirmed in the future study by more epidemiological investigations of human C. sinensis infection from different areas.

Haplotype comparisons of Echinococcus granulosus sensu lato via mitochondrial gene sequences (co1, cytb, nadh1) among Pakistan and its neighbouring countries.

Echinococcus granulosus sensu lato (s.l.) is a zoonotic parasite that causes cystic echinococcosis (CE) in humans. However, E. granulosus sensu stricto (s.s.) is considered the predominant species in CE infections worldwide. According to the population genetic diversity and structure of E. granulosus s.l., gene flow can explain the parasite drift among the neighbouring countries of Pakistan. The mitochondrial (mt) co1 (n = 47), nadh1 (n = 37) and cytb (n = 35) nucleotide sequences of E. granulosus s.l. isolates from Pakistan, Iran, China and India were retrieved from the National Centre for Biotechnology Information database to determine the genealogical relationships. The sequences were grouped as the mt-co1 (genotypes G1 and G3, G6-G7), mt-cytb (genotypes G1 and G3), and mt-nadh1(genotypes G1 and G3). The data were analysed using bioinformatic tools. A total of 19 polymorphic sites for the mt-co1 sequence (374 bp) were observed of which 31.6% (6/19) were parsimony-informative sites. Unique singleton haplotypes within the E. granulosus s.s. haplotype network based on the mt-co1 gene were highly prevalent (68.4%; 13/19) in Pakistani isolates followed by Chinese, Indian and Iranian isolates; four polymorphic sites were detected in the E. canadensis (G6/G7). In E. canadensis mt-co1 haplotype network, 75% (3/4) unique singleton haplotypes were from the Iranian isolates. Twelve polymorphic sites were found using the mt-cytb sequence (547 bp); 25% (3/12) were parsimony-informative and there were 66.7% (8/12) unique singleton haplotypes within the mt-cytb haplotype network in E. granulosus s.s. with the most reported from Pakistan followed by Iran and China. 20 polymorphic sites were detected in E. granulosus s.s. mt-nadh1 sequences (743 bp); 20% (4/20) were parsimony-informative. There were 66.7% (8/12) main single haplotypes within the mt-nadh1 haplotype network, with the most reported from Pakistan followed by that from India, Iran and China. The sequence analyses show low nucleotide diversity and high haplotype diversity in general.

Identification of Different Extracellular Vesicles in the Hydatid Fluid of Echinococcus granulosus and Immunomodulatory Effects of 110 K EVs on Sheep PBMCs.

Echinococcosis, mainly caused by Echinococcus granulosus, is one of the 17 neglected tropical diseases. Extracellular vesicles (EVs) play an essential role in the host-parasite interplay. However, the EVs in the hydatid fluid (HF) of E. granulosus are not fully characterized. Herein, three different types of HF EVs, designated as 2 K, 10 K, and 110 K EVs based on the centrifugal force used, were morphologically identified. A total of 97, 80, and 581 proteins were identified in 2 K, 10 K, and 110 K EVs, respectively, 39 of which were commonly shared. Moreover, 11, 8, and 25 miRNAs were detected, respectively, and all of the 7 selected miRNAs were validated by qPCR to be significantly lower abundant than that in protoscoleces. It was further deemed that 110 K EVs were internalized by sheep peripheral blood mononuclear cells (PBMCs) in a time-dependent manner and thus induced interleukin (IL)-10, tumor necrosis factor-α (TNF-α), and IRF5 were significantly upregulated and IL-1ß, IL-17, and CD14 were significantly downregulated (p < 0.05). These data demonstrate the physical discrepancy of three HF EVs and an immunomodulatory effect of 110 K EVs on sheep PMBCs, suggesting a role in immune responses during E. granulosus infection.

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model.

BACKGROUND: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. METHODS: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. RESULTS: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 µg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. CONCLUSIONS: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Identification of Uncommon Cryptosporidiumviatorum (a Novel Subtype XVcA2G1c) and Cryptosporidium andersoni as Well as Common Giardia duodenalis Assemblages A and B in Humans in Myanmar.

Cryptosporidium and Giardia are two important zoonotic intestinal protozoa responsible for diarrheal diseases in humans and animals worldwide. Feces from infected hosts, water and food contaminated by Cryptosporidium oocysts and Giardia cysts as well as predictors such as poverty have been involved in their transmission. Myanmar is one of the world's most impoverished countries. To date, there are few epidemiological studies of Cryptosporidium and Giardia in humans. To understand the prevalence and genetic characterization of Cryptosporidium spp. and Giardia duodenalis in humans in Myanmar, a molecular epidemiological investigation of the two protozoa was conducted in four villages of Shan State. 172 fecal specimens were collected from Wa people (one each) and identified for the presence of Cryptosporidium spp. and G. duodenalis by sequence analysis of their respective small subunit ribosomal RNA genes. 1.74% of investigated people were infected with Cryptosporidium spp.-C. andersoni (n = 2) and C. viatorum (n = 1) while 11.05% infected with G. duodenalis-assemblages A (n = 6) and B (n = 13). By sequence analysis of 60-kDa glycoprotein gene, the C. viatorum isolate belonged to a novel subtype XVcA2G1c. DNA preparations positive for G. duodenalis were further subtyped. Five of them were amplified and sequenced successfully: different assemblage B sequences (n = 2) at the triosephosphate isomerase (tpi) locus; sub-assemblage AII sequence (n = 1) and identical assemblage B sequences (n = 2) at the ß-giardin (bg) locus. This is the first molecular epidemiological study of Cryptosporidium spp. and G. duodenalis in humans in Myanmar at both genotype and subtype levels. Due to unclear transmission patterns and dynamics of Cryptosporidium spp. and G. duodenalis, future research effort should focus on molecular epidemiological investigations of the two parasites in humans and animals living in close contact in the investigated areas, even in whole Myanmar. These data will aid in making efficient control strategies to intervene with and prevent occurrence of both diseases.

Comprehensive Analysis of Non-coding RNA Profiles of Exosome-Like Vesicles From the Protoscoleces and Hydatid Cyst Fluid of Echinococcus granulosus.

Cystic echinococcosis is a worldwide chronic zoonotic disease that threatens human health and animal husbandry. Exosome-like vesicles (ELVs) have emerged recently as mediators in the parasite-parasite intercommunication and parasite-host interactions. Exosome-like vesicles from parasites can transfer non-coding RNAs (ncRNAs) into host cells to regulate their gene expression; however, the ncRNAs profiles of the ELVs from Echinococcus granulosus remain unknown. Here, we isolated protoscolece (PSC)-ELVs and hydatid fluid (HF)-ELVs from the culture medium for E. granulosus PSCs in vitro and the HF of fertile sheep cysts, respectively. The microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) profiles of the two types of ELVs were analyzed using high-throughput sequencing, and their functions were predicted using Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis. In PSC-ELVs and HF-ELVs, 118 and 58 miRNAs were identified, respectively, among which 53 miRNAs were present in both ELVs, whereas 65 and 5 miRNAs were unique to PSC-ELVs and HF-ELVs, respectively; 2,361 and 1,254 lncRNAs were identified in PSC-ELVs and HF-ELVs, respectively, among which 1,004 lncRNAs were present in both ELVs, whereas 1,357 and 250 lncRNAs were unique to PSC-ELVs and HF-ELVs, respectively. Intriguingly, the spilled PSCs from cysts excrete ELVs with higher numbers of and higher expression levels of miRNAs and circRNAs than HF-ELVs. The miRNA sequencing data were validated by quantitative reverse transcription-polymerase chain reaction. Furthermore, the target lncRNAs and mRNAs regulated by the 20 most abundant miRNAs were screened, and a ceRNA regulatory network containing 5 miRNAs, 41 lncRNAs, and 23 mRNAs was constructed, which provided new ideas and the molecular basis for further clarification of the function and mechanism of E. granulosus ELVs ncRNAs in the parasite-host interactions. Egr-miR-125-5p and egr-miR-10a-5p, sharing identical seed sites with host miRNAs, were predicted to mediate inflammatory response, collagen catabolic process, and mitogen-activated protein kinase cascade during parasite infections. In conclusion, for the first time, we identified the ncRNAs profiles in PSC-ELVs and HF-ELVs that might be involved in host immunity and pathogenesis, and enriched the ncRNAs data of E. granulosus. These results provided valuable resources for further analysis of the regulatory potential of ncRNAs, especially miRNAs, in both types of ELVs at the parasite-host interface.

IL-17A-producing γδ T cells promote liver pathology in acute murine schistosomiasis.

BACKGROUND: The main symptoms of schistosomiasis are granuloma and fibrosis, caused by Schistosoma eggs. Numerous types of cells and cytokines are involved in the progression of Schistosoma infection. As a class of innate immune cells, γδ T cells play critical roles in the early immune response. However, their role in modulating granuloma and fibrosis remains to be clarified. METHODS: Liver fibrosis in wild-type (WT) mice and T cell receptor (TCR) δ knockout (KO) mice infected with Schistosoma japonicum was examined via Masson's trichrome staining of collagen deposition and quantitative reverse transcriptase-PCR (RT-PCR) of fibrosis-related genes. Granuloma was detected by hematoxylin-eosin (H&E) staining and quantified. Flow cytometry was used for immune cell profiling and for detecting cytokine secretion. The abundance of the related cytokines was measured using quantitative RT-PCR. RESULTS: The livers of S. japonicum-infected mice had significantly increased proportions of interleukin (IL)-17A producing γδ T cells and secreted IL-17A. Compared with the WT mice, TCR δ deficiency resulted in reduced pathological impairment and fibrosis in the liver and increased survival in infected mice. In addition, the profibrogenic effects of γδ T cells in infected mice were associated with enhanced CD11b+Gr-1+ cells, concurrent with increased expression of transforming growth factor (TGF)-ß in the liver. CONCLUSIONS: In this mouse model of Schistosoma infection, γδ T cells may promote liver fibrosis by recruiting CD11b+Gr-1+ cells. These findings shed new light on the pathogenesis of liver pathology in murine schistosomiasis.

Superparamagnetic Nanostructures for Split-Type and Competitive-Mode Photoelectrochemical Aptasensing.

Photoelectrochemical sensing has developed rapidly in the past decade because of its inherent advantages of economic devices and low background noise. However, traditional assembly of photoelectric beacons, probes, and targets on the ITO electrode solid-liquid interface inevitably leads to time-consuming, limited selectivity, poor stability, and nonreproducibility. To overcome these drawbacks, in this work, a unique split-type PEC aptasensor for carcinoembryonic antigen (CEA) was developed in virtue of the sandwich-like structure comprised of magnetic-optical Fe3O4@SiO2@CdS-DNA1, CEA aptamer, and signal element SiO2-Au-DNA2. The sandwich-like structure is easily formed in the liquid phase and can be triggered by competition from low-abundance CEA, resulting in dissociation. By further photocurrent measurement in pure phosphate buffer saline (PBS), coexisting species can be effectively removed from the modified electrode, improving selectivity, stability, and repeatability. These advantages benefit from the preparation of uniform and monodispersed Fe3O4@SiO2@CdS and SiO2-Au particles, DNAs assembly, and an elegant design. Additionally, the as-designed signal-on PEC aptasensor is highly sensitive, short time-consuming, and economical, enabling the detection of CEA in serum specimens. It not only provides an alternative to CEA immunosensors, but also paves the way for high-performance PEC aptasensors.

Molecular characterization of human Echinococcus isolates and the first report of E. canadensis (G6/G7) and E. multilocularis from the Punjab Province of Pakistan using sequence analysis.

BACKGROUND: Echinococcosis is a zoonotic parasitic disease causing serious health problems in both humans and animals in different endemic regions across the world. There are two different forms of human echinococcosis: Cystic Echinococcosis (CE) and Alveolar Echinococcosis (AE). CE is caused by the larval stage of Echinococcus granulosus sensu lato and AE by the larval stage of Echinococcus multilocularis. Geographically, CE is universally distributed, while AE is prevalent in the northern hemisphere. Although the disease is endemic in neighboring countries (China, Iran and India) of Pakistan, there are limited reports from that country. Besides, there are no comprehensive data on the genotyping of Echinococcus species in humans based on sequence analysis. This study aimed to detect the presence of human CE and to identify Echinococcus spp. in human isolates through genetic characterization of hydatid cysts in the Punjab Province of Pakistan. METHODS: Genetic analysis was performed on 38 human hydatid cyst samples collected from patients with echinococcosis using mitochondrial cytochrome c oxidase subunit 1 (cox1), cytochrome b (cytb) and NADH subunit 1 (nad1). Patient data including age, epidemiological history, sex, and location were obtained from hospital records. RESULTS: According to the sequence analysis we detected E. granulosus sensu stricto (n = 35), E. canadensis (G6/G7) (n = 2), and E. multilocularis (n = 1). Thus, the majority of the patients (92.1%, 35/38) were infected with E. granulosus s.s. This is the first molecular confirmation of E. canadensis (G6/G7) and E. multilocularis in human subjects from Pakistan. CONCLUSIONS: These findings suggested that E. granulosus s.s. is the dominant species in humans in Pakistan. In addition, E. canadensis (G6/G7) and E. multilocularis are circulating in the country. Further studies are required to explore the genetic diversity in both humans and livestock.

Arginase promotes immune evasion of Echinococcus granulosus in mice.

BACKGROUND: Cystic echinococcosis is a chronic disease caused by infection with the larvae of Echinococcus granulosus. The parasite's ability to establish persistent infection is partly due to its evolving immune evasion strategies. One strategy may involve the protective effect of arginase, which impedes the control of pathogens or tumors, whereas it remains largely unknown during E. granulosus infection. Here, we analyzed whether arginase was produced in peritoneal cells and assessed its role in immunosuppression in mice infected with protoscoleces of E. granulosus. METHODS: BALB/c mice injected with protoscoleces of E. granulosus were used to evaluate the expression of arginase (ARG) in mRNA and protein levels. The profiles of ARG-1 expression in peritoneal cells and CD3ζ expression in T cells from spleens were assessed at different time points (3, 6, 9 and 12 months post-infection) by flow cytometry. In vitro, peritoneal cells were co-cultured with purified T cells in a transwell system, and the levels of CD3ζ re-expression were compared by flow cytometry. Meanwhile, the changes of L-arginine and its related metabolites in serum were tested. RESULTS: Compared to the control group, the peritoneal cells from infected mice showed higher levels of ARG-1 mRNA and protein, unchanged ARG-2 and iNOS. Enhanced ARG-1 expression was present in SSClowCD11b+F4/80+, CD11b+CD11c+, CD11b+Gr-1+Ly-6C+Ly-6G-, CD11b+Gr-1+Ly-6C-Ly-6G+, CD11b+Gr-1+ and CD11b+Ly-6G+ cells. The proportion of cells and the proportion of ARG-1 expression in corresponding cells exhibited a rising trend along with the extension of infection time, except for fluctuations in SSClowCD11b+F4/80+ and CD11b+CD11c+ cells at 12 months post-infection, whereas the expression of CD3ζ chain in CD4+ and CD8+ T cells showed a descending trend. Purified T cells showed declined re-expression of CD3ζ when co-cultured with peritoneal cells from infected mice, and CD3ζ was regenerated by supplement of L-arginine or arginase inhibitor BEC, rather than NOS inhibitor L-NMMA or catalase. Meanwhile, the concentrations of L-arginine, L-citrulline and NO decreased, and those of L-ornithine and urea increased in serum post-infection. CONCLUSIONS: Our findings demonstrated that ARG-1 expression is enhanced in multiple myeloid cells from peritoneum and promotes immune evasion of E. granulosus in mice by inhibiting the expression of T cell receptor CD3ζ chain and antagonism against iNOS.