Oral Delivery of Anti-Parasitic Agent-Loaded PLGA Nanoparticles: Enhanced Liver Targeting and Improved Therapeutic Effect on Hepatic Alveolar Echinococcosis.

Background: Alveolar echinococcosis (AE) is a lethal parasitic disease caused by infection with the metacestode of the dog/fox tapeworm Echinococcus multilocularis, which primarily affects the liver. Although continued efforts have been made to find new drugs against this orphan and neglected disease, the current treatment options remain limited, with drug delivery considered a likely barrier for successful treatment. Methods: Nanoparticles (NPs) have gained much attention in the field of drug delivery due to their potential to improve delivery efficiency and targetability. In this study, biocompatible PLGA nanoparticles encapsulating a novel carbazole aminoalcohol anti-AE agent (H1402) were prepared to promote the delivery of the parent drug to liver tissue for treating hepatic AE. Results: H1402-loaded nanoparticles (H1402-NPs) had a uniform spherical shape and a mean particle size of 55 nm. Compound H1402 was efficiently encapsulated into PLGA NPs with a maximal encapsulation efficiency of 82.1% and drug loading content of 8.2%. An in vitro uptake assay demonstrated that H1402-NPs rapidly penetrated the in vitro cultured pre-cyst wall and extensively accumulated in the pre-cysts of E. multilocularis within only 1 h. The biodistribution profile of H1402-NPs determined through ex vivo fluorescence imaging revealed significantly enhanced liver distribution compared to unencapsulated H1402, which translated to improved therapeutic efficacy and reduced systemic toxicity (especially hepatotoxicity and cytotoxicity) in a hepatic AE murine model. Following a 30-day oral regimen (100 mg/kg/day), H1402-NPs significantly reduced the parasitic burden in both the parasite mass (liver and metacestode total weight, 8.8%) and average metacestode size (89.9%) compared to unmedicated infected mice (both p-values < 0.05); the treatment outcome was more effective than those of albendazole- and free H1402-treated individuals. Conclusion: Our findings demonstrate the advantages of encapsulating H1402 into PLGA nanoparticles and highlight the potential of H1402-NPs as a promising liver-targeting therapeutic strategy for hepatic AE.

A Diverse Virome Is Identified in Parasitic Flatworms of Domestic Animals in Xinjiang, China.

Parasitic flatworms infect diverse vertebrates and are major threats to animal and even human health; however, little is known about the virome of these lower life forms. Using viral metagenomic sequencing, we characterized the virome of the parasitic flatworms collected from major domestic animals, including Dicrocoelium lanceatum and Taenia hydatigena, Echinococcus granulosus sensu stricto and Echinococcus multilocularis. Seven and three different viruses were discovered from D. lanceatum and T. hydatigena, respectively, and no viral sequences were found in adult tapeworms and protoscoleces of E. granulosus sensu stricto and E. multilocularis. Two out of the five parasitic flatworm species carry viruses, showing a host specificity of these viruses. These viruses belong to the Parvoviridae, Circoviridae, unclassified circular, Rep-encoding single-stranded (CRESS) DNA virus, Rhabdoviridae, Endornaviridae, and unclassified RNA viruses. The presence of multiple highly divergent RNA viruses, especially those that cluster with viruses found in marine animals, implies a deep evolutionary history of parasite-associated viruses. In addition, we found viruses with high identity to common pathogens in dogs, including canine circovirus and canine parvovirus 2. The presence of these viruses in the parasites implies that they may infect parasitic flatworms but does not completely exclude the possibility of contamination from host intestinal contents. Furthermore, we demonstrated that certain viruses, such as CRESS DNA virus may integrate into the genome of their host. Our results expand the knowledge of viral diversity in parasites of important domestic animals, highlighting the need for further investigations of their prevalence among other parasites of key animals. IMPORTANCE Characterizing the virome of parasites is important for unveiling the viral diversity, evolution, and ecology and will help to understand the "Russian doll" pattern among viruses, parasites, and host animals. Our data indicate that diverse viruses are present in specific parasitic flatworms, including viruses that may have an ancient evolutionary history and viruses currently circulating in parasite-infected host animals. These data also raise the question of whether parasitic flatworms acquire and/or carry some viruses that may have transmission potential to animals. In addition, through the study of virus-parasite-host interactions, including the influence of viral infection on the life cycle of the parasite, as well as its fitness and pathogenicity to the host, we could find new strategies to prevent and control parasitic diseases.

Echinococcus granulosus sensu stricto and antigen B may decrease inflammatory bowel disease through regulation of M1/2 polarization.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic idiopathic disease characterized by inflammation-related epithelial barrier damage in the intestinal tract. Helminth infection reduces autoimmune disease symptoms through regulation of inflammatory responses based on hygiene theory. However, the underlying mechanisms remain unclear. METHODS: BALB/c mice were infected with microcysts of E. granulosus sensu stricto and drank water containing 3.5% dextran sodium sulfate (DSS) at the 100th day post-infection. After 7 days of drinking DSS, the mouse body weight change and disease activity index (DAI) were recorded every day, and colon length and histological score were evaluated after sacrifice. After injection with antigen B (AgB), inducible nitric oxide synthase (iNOS) and Fizz1 expression and F4/80+CD11c+ M1 and F4/80+CD206+ M2 in the peritoneal cells and colon tissues were analysed by qPCR and flow cytometry, respectively. Gut microbiota were profiled by 16S rRNA sequencing of the mouse faecal samples. For in vitro assay, RAW264.7 macrophages were cultured in medium containing AgB before induction by lipopolysaccharide (LPS). Then, NO in the supernatant was measured, and the expression of cytokine genes associated with macrophages were determined by qRT-PCR. RESULTS: Echinococcus granulosus s.s. infection and AgB significantly reduced the symptoms and histological scores of IBD induced by DSS (P < 0.05). Flow cytometry showed that AgB inoculation increased F4/80+ and CD206+ in peritoneal cells. The results of qPCR showed that AgB significantly decreased iNOS and increased Fizz1 expression in the colon of mice inoculated by DSS (P < 0.05). Furthermore, AgB injection led to significant changes in the profiles of five genera (Paraprevotella, Odoribacter, Clostridium cluster XlVa, Oscillibacter, and Flavonifractor) in faecal samples. In vitro analysis showed that AgB reduced NO levels (P < 0.01), with a significant decrease in iNOS expression (P < 0.05) in RAW264.7 cells induced by LPS. CONCLUSIONS: Echinococcus granulosus infection and AgB may improve IBD conditions by inducing an M2-predominant cellular (F4/80+ CD206+) profile and decreasing type 1 macrophages (F4/80+CD11c+) in the intestinal lamina propria. In addition, AgB intervention induced changes in the microbiota condition of the gastrointestinal duct and reversed NO expression. Thus, AgB may be a drug candidate for IBD treatment.

In vitro Scolicidal Efficacy of 5-Fluorouracil and Radiation Against Protoscoleces of Echinococcus granulosus Sensu Lato.

PURPOSE: Cystic echinococcosis (CE) caused by Echinococcus granulosus sensu lato (s.l.) is a globally distributed zoonosis. CE treatment is difficult, but radiation and 5-fluorouracil (5-FU) can be effective. However, the combination of radiation and 5-FU has not been reported. This study evaluated the effect of combination of 5-FU and radiation on E. granulosus s.l. protoscoleces (PSCs). MATERIAL AND METHODS: In this study, PSCs were collected from the liver of diseased sheep, and some were exposed to a single dose of 20 Gy 6-MV X-ray combined with (5 µg/mL or 10 µg/mL) 5-FU in vitro. Methylene blue staining was used to detect the viability of the PSCs. Transcription of EgHSP70 and Egp38 was measured by quantitative real-time PCR (qRT- PCR). RESULTS: A single dose of radiation killed 18% of the PSCs, and 5-FU showed weak parasiticidal efficacy on the first day of treatment. After 14 d, 5 µg and 10 µg/mL of 5-FU killed 40.20% and 50.02% of the PSCs, whereas 20 Gy of radiation killed 31.44%. The combination of 5-FU (10 µg/mL) with 20 Gy of radiation showed 77.55% killing efficacy. qRT-PCR showed that 5-FU inhibited Egp38 expression, whereas radiation increased its expression. EgHSP70 was highly expressed 14 days after radiation treatment. The data indicate that 5-FU has parasiticidal efficacy against the PSCs of E. granulosus s.l. CONCLUSION: The lethal efficacy of PSCs caused by a single dose of radiation exposure is related to the upregulated expression level of Egp38 and EgHSP70. The killing effect of 5-FU (10 µg/mL) with 20Gy of radiation was significantly better than that of single treatment group. This study provided a basis for the potential role of 5-FU combined with radiation in the treatment of CE.

Adaptive Latent Graph Representation Learning for Image-Text Matching.

Image-text matching is a challenging task due to the modality gap. Many recent methods focus on modeling entity relationships to learn a common embedding space of image and text. However, these methods suffer from distractions of entity relationships such as irrelevant visual regions in an image and noisy textual words in a text. In this paper, we propose an adaptive latent graph representation learning method to reduce the distractions of entity relationships for image-text matching. Specifically, we use an improved graph variational autoencoder to separate the distracting factors and latent factor of relationships and jointly learn latent textual graph representations, latent visual graph representations, and a visual-textual graph embedding space. We also introduce an adaptive cross-attention mechanism to perform feature attending on the latent graph representations across images and texts, thus further narrowing the modality gap to boost the matching performance. Extensive experiments on two public datasets, Flickr30K and COCO, show the effectiveness of our method.

Bioinformatic comparison of Kunitz protease inhibitors in Echinococcus granulosus sensu stricto and E. multilocularis and the genes expressed in different developmental stages of E. granulosus s.s.

BACKGROUND: Cystic and alveolar echinococcosis caused by the tapeworms Echinococcus granulosus sensu stricto (s.s.) and E. multilocularis, respectively, are important zoonotic diseases. Protease inhibitors are crucial for the survival of both Echinococcus spp. Kunitz-type inhibitors play a regulatory role in the control of protease activity. In this study,we identified Kunitz-type domain protease inhibitors(KDPIs) present in the genomes of these two tapeworms and analyzed the gene sequences using computational, structural bioinformatics and phylogenetic approaches to evaluate the evolutionary relationships of these genes. Hi-seq transcriptome analysis showed that E. granulosus s.s. KDPIs were differentially expressed in the different developmental stages. We validated some of the genes expressed in adult worm, protoscolex and cyst germinal membrane of E. granulosus s.s. and E. multilocularis by quantitative PCR. RESULTS: A total of 19 genes from E. multilocularis and 23 genes from E. granulosus s.s. were predicted to be KDPIs with the most containing a single Kunitz-domain. A maximum likelihood method phylogenetic tree indicated that the E. granulosus s.s. and E. multilocularis Kunitz domain peptides were divided into three branches containing 9 clusters. The ratio of positively charged residues and neutral residues are different between E. multilocularis and E. granulosus s.s. KDPIs. We also found that E. multilocularis had higher percentage of sequences containing signal peptides (17/19, 89.47%) than that of E. granulosus s.s. (14/23, 60.87%). Transcript analysis showed all the E. granulosus s.s. KDPI genes were expressed differentially in four developmental stages of the worm. Transcription analysis showed that 9 KDPIs (including EG_07244,EGR_08716 and EGR_10096) were highly upregulated in adult worm, and 2 KDPIs (EG_09268 and EG_09490) were highly expressed in the cyst germinal membrane. Quantitative gene expression analysis(qPCR) of four genes confirmed the expression of these genes. EGR_08716 and its homologous gene (EmuJ_001137000) were highly and specifically expressed in adult worms of the two worms. CONCLUSIONS: A total 19 and 23 KDPIs were identified in the genomes of E. multilocularis and E. granulosus s.s. , respectively. The differential expression of these KDPIs in different stages may indicate their different roles in the different hosts. The difference in characterization of KDPIs may be associated with the different pathology of metacestode stage of these two parasites.

High endemicity of alveolar echinococcosis in Yili Prefecture, Xinjiang Autonomous Region, the People's Republic of China: Infection status in different ethnic communities and in small mammals.

Alveolar echinococcosis (AE) is a life-threatening disease in humans caused by the larval stage of Echinococcus multilocularis. The tapeworm is transmitted between small mammals and dogs/foxes in the Northern Hemisphere. In this study 286 AE cases were reported from eight counties and one city in Yili Prefecture, Xinjiang Autonomous Region, the People's Republic of China from 1989 to 2015 with an annual incidence (AI) of 0.41/100,000. Among the patients, 73.08% were diagnosed in the last 11 years. Four counties in the high mountainous areas showed higher AI (0.51-1.22 cases/100,000 residents) than the four counties in low level areas (0.19-0.29/100,000 residents). The AI of AE in Mongolian (2.06/100,000 residents) and Kazak (0.93/100,000 residents) ethnic groups was higher than the incidence in other ethnic groups indicating sheep-farming is a risk for infection given this activity is mainly practiced by these two groups in the prefecture. A total of 1411 small mammals were captured with 9.14% infected with E. multilocularis metacestodes. Microtus obscurus was the dominant species in the mountain pasture areas with 15.01% of the voles infected, whereas Mus musculus and Apodemus sylvaticus were the dominant small mammals in the low altitude areas. Only 0.40% of A. sylvaticus were infected with E. multilocularis. PCR amplification and sequencing analysis of the mitochondrial cox1 gene showed that E. multilocularis DNA sequences from the small mammals were identical to isolates of local human AE cases. The overall results show that Yili Prefecture is a highly endemic area for AE and that the high-altitude pasture areas favorable for M. obscurus may play an important role in its transmission in this region.