Adult pancreatoblastoma with atypical histological morphology combined with familial adenomatous polyposis: a rare case report.

Pancreatoblastoma (PB) is a rare malignant pancreatic epithelial tumor that mostly occurs in children and occasionally occurs in adults. The tumor has acinar cell differentiation and squamous corpuscles/squamous epithelial islands, which are frequently separated by fibrous bundles. Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by the presence of numerous adenomatous polyps in the colon and rectum. Cases of pancreatoblastoma combined with familial adenomatous polyposis (FAP) are rarely reported. A review of a rare case of adult pancreatoblastoma with atypical histological morphology combined with familial adenomatous polyposis is presented herein. In this case, the patient was first diagnosed with familial adenomatous polyposis and subsequently found to have pancreatoblastoma 1 year and 3 months later. This suggests pancreatoblastoma may occur in patients with familial adenomatous polyposis or a family history of the condition, indicating a possible association between the two tumors. Therefore, pancreatoblastoma should be included in a differential diagnosis for FAP patients with a pancreatic mass. The final diagnosis of pancreatoblastoma depends on the pathological diagnosis. Acinar-like cells and squamous corpuscles/squamous epithelial cell islands under light microscopy are the key diagnostic points. This case report also can improve the awareness of clinicians, radiologists, and pathologists on the presence of rare tumor-adult pancreatoblastoma in patients with familial adenomatous polyposis.

Molecular characterization and immunological properties of Echinococcus granulosus sensu stricto (G1) ADK1 and ADK8.

Adenylate kinases (ADKs) are one of the important enzymes regulating adenosine triphosphate (ATP) metabolism in Echinococcus granulosus sensu lato. The objective of the present study was to explore the molecular characteristics and immunological properties of E. granulosus sensu stricto (G1) adenylate kinase 1 (EgADK1) and adenylate kinase 8 (EgADK8). EgADK1 and EgADK8 were cloned and expressed, and the molecular characteristics of EgADK1 and EgADK8 were analyzed through different bioinformatics tools. Western blotting was used to examine the reactogenicity of recombinant adenylate kinase 1 (rEgADK1) and recombinant adenylate kinase 8 (rEgADK8) and to evaluate their diagnostic value. The expression profiles of EgADK1 and EgADK8 in 18-day-old strobilated worms and protoscoleces were analyzed by quantitative real-time PCR, and their distribution in 18-day-old strobilated worms, the germinal layer, and protoscoleces was determined by immunofluorescence localization. EgADK1 and EgADK8 were successfully cloned and expressed. Bioinformatics analysis predicted that EgADK1 and EgADK8 have multiple phosphorylation sites and B-cell epitopes. Compared with EgADK8, EgADK1 and other parasite ADKs have higher sequence similarity. In addition, both cystic echinococcosis (CE)-positive sheep sera and Cysticercus tenuicollis-infected goat sera could recognize rEgADK1 and rEgADK8. EgADK1 and EgADK8 were localized in protoscoleces, the germinal layer, and 18-day-old strobilated worms. EgADK1 and EgADK8 showed no significant difference in their transcription level in 18-day-old strobilated worms and protoscoleces, suggesting that EgADK1 and EgADK8 may play an important role in the growth and development of E. granulosus sensu lato. Since EgADK1 and EgADK8 can be recognized by other parasite-positive sera, they are not suitable as candidate antigens for the diagnosis of CE.

Macrophage Migration Inhibitory Factor in Psoroptes ovis: Molecular Characterization and Potential Role in Eosinophil Accumulation of Skin in Rabbit and Its Implication in the Host-Parasite Interaction.

Psoroptes ovis, a common surface-living mite of domestic and wild animals worldwide, results in huge economic losses and serious welfare issues in the animal industry. P. ovis infestation rapidly causes massive eosinophil infiltration in skin lesions, and increasing research revealed that eosinophils might play an important role in the pathogenesis of P. ovis infestation. Intradermal injection of P. ovis antigen invoked massive eosinophil infiltration, suggesting that this mite should contain some relative molecules involved in eosinophil accumulation in the skin. However, these active molecules have not yet been identified. Herein, we identified macrophage migration inhibitor factor (MIF) in P. ovis (PsoMIF) using bioinformatics and molecular biology methods. Sequence analyses revealed that PsoMIF appeared with high similarity to the topology of monomer and trimer formation with host MIF (RMSD = 0.28 angstroms and 2.826 angstroms, respectively) but with differences in tautomerase and thiol-protein oxidoreductase active sites. Reverse transcription PCR analysis (qRT-PCR) results showed that PsoMIF was expressed throughout all the developmental stages of P. ovis, particularly with the highest expression in female mites. Immunolocalization revealed that MIF protein located in the ovary and oviduct of female mites and also localized throughout the stratum spinosum, stratum granulosum, and even basal layers of the epidermis in skin lesions caused by P. ovis. rPsoMIF significantly upregulated eosinophil-related gene expression both in vitro (PBMC: CCL5, CCL11; HaCaT: IL-3, IL-4, IL-5, CCL5, CCL11) and in vivo (rabbit: IL-5, CCL5, CCL11, P-selectin, ICAM-1). Moreover, rPsoMIF could induce cutaneous eosinophil accumulation in a rabbit model and increased the vascular permeability in a mouse model. Our findings indicated that PsoMIF served as one of the key molecules contributing to skin eosinophil accumulation in P. ovis infection of rabbits.

Solitary Fibrous Tumors of the Lung: A Clinicopathological Analysis of 52 Cases.

OBJECTIVE: To explore the clinicopathological features of solitary fibrous tumors (SFTs) of the lung. METHODS: We collected the clinical data of 52 patients with SFTs of the lung confirmed by pathology, and summarized the clinical, radiological, and morphological features, the immunophenotypes, and the prognosis of SFTs. RESULTS: Fifty-two cases of SFTs of the lung were enrolled in this study, including 51 cases of borderline and 1 case of malignancy, 22 males and 30 females. The average onset age was 52.7 years. The lower lobe of the left lung was the preferred site of SFTs, accounting for 30.77% (16/52). Chest CT showed regular and well-demarcated soft tissue density mass, and the tumor size of most cases (46/52, 88.46%) was 1-10 cm. Morphological features: The distribution of tumor cells showed sparse and dense areas. Tumor cells were spindle-shaped, in whorls or hemangiopericytoma-like conformation. Atypia, mitotic figures, and necrosis were found. Immunohistochemistry showed positive expression of CD34, STAT6, Vimentin, BCL2, and CD99. Ki-67 was ≤ 5% in borderline SFTs and >20% in a malignant SFT. CONCLUSIONS: Solitary fibrous tumors of the lung occur more frequently in middle-aged and elderly people, and there is no significant difference in gender. The lower lobe of the left lung is the preferred site of SFTs. The size of most SFTs is 1-10 cm. Chest CT shows morphologically regular and well-demarcated soft tissue density mass. Pathologically, SFTs of the lung are mostly borderline and occasionally malignant. Immunohistochemistry shows the positive expression of CD34, STAT6, Vimentin, BCL2, and CD99.

Artificial Infestation of Sarcoptes scabiei (Acari: Sarcoptidae) in Rabbits Exhibits Progressive Pathological Changes, Apoptosis, and Keratinization in the Skin.

Sarcoptes scabiei (S. scabiei) is an ectoparasite that can infest humans and 150 mammalian host species, primarily causing pruritus, crust, and alopecia. However, neither the pathological process of host skin under S. scabiei infection nor the mechanism of S. scabiei infection in regulating apoptosis and keratinization of host skin has been studied yet. In this study, a total of 56 rabbits were artificially infested with S. scabiei, and the skin samples were collected at seven different time points, including 6 h, 12 h, day 1, day 3, 1 week, 4 weeks, and 8 weeks, whereas a group of eight rabbits served as controls. We measured epidermal thickness by H&E staining, observed the skin ultrastructure by electron microscopy, and detected the degree of skin apoptosis by TUNEL staining. The level of transcription of genes related to apoptosis and keratinization was detected by quantitative real-time PCR (qRT-PCR), and the level of Bcl-2 protein expression was further detected using immunohistochemistry. Our results showed that, with increased infestation time, the epidermal layer of the rabbit skin exhibited significant thickening and keratinization, swollen mitochondria in the epidermal cells, and increased skin apoptosis. The level of caspase-1, 3, 8, 10, 14, and Bcl-2 mRNA expression was increased, whereas the level of keratin 1 and 5 was decreased after S. scabiei infestation. In conclusion, S. scabiei infestation causes thickening of the epidermis, which may be related to apoptosis-induced proliferation and skin keratinization.

Vaccination with a cocktail vaccine elicits significant protection against Sarcoptes scabiei in rabbits, whereas the multi-epitope vaccine offers limited protection.

Sarcoptes scabiei cause scabies in humans or sarcoptic mange in animals. Currently, information regarding vaccines against S. scabiei is limited and no commercial vaccine is available. In present study, we expressed and mixed recombinant S. scabiei serpin (rSs-serpin), recombinant S. scabiei chitinase-like protein-5 [rSs-CLP5] and -12 [rSs-CLP12] as a cocktail vaccine (three proteins mixed), and also a multi-epitope protein derived from these three S. scabiei genes was expressed as a vaccine candidate to evaluate the effects of two vaccine strategies. Four test groups (n = 12 per group) and a control group (n = 12 per group) were involved in this vaccination trial. The results showed that 91.67% (11/12) and 83.33% (10/12) of rabbits exhibited no detectable skin lesions from S. scabiei infestation in cocktail vaccine groups, whereas two multi-epitope groups produced only a few rabbits (5/12, 6/12) having no detectable skin lesions. Four test groups displayed significant increases in specific IgG antibodies (Abs) and total IgE Abs after immunized with recombinant proteins. Taken together, our data demonstrated a mixture of rSs-serpin, rSs-CLP5 and rSs-CLP12 was a promising vaccine candidate that induced robust immune protection and could significantly decrease mite populations to reduce the direct transmission between rabbits. However, vaccination with the multi-epitope protein showed limited protection in rabbits.

Evaluation of the immune protective effects of rEmMIC2 and rEmMIC3 from Eimeria magna in rabbits.

Eimeria magna is a common pathogen in rabbits, which results in lethargy, weight loss, diarrhea, and even death in severe cases after infection. The current method for preventing rabbit coccidiosis is to add anticoccidial drugs to the diet. However, there are many concerns about drug resistance and drug residues. In our study, the rEmMIC2 and rEmMIC3 proteins were cloned and expressed to evaluate potential as recombinant subunit vaccine candidate antigens. The protective effects of rEmMIC2 and rEmMIC3 were evaluated by the relative weight gain ratio, oocyst decrease rate, anticoccidial index, feed conversion ratio, pathological alterations, clinical symptoms, specific IgG antibody, and cytokine levels in rabbits. The molecular weights of rEmMIC2 and rEmMIC3 were 18.69 kDa and 17.47 kDa, respectively. After the coccidia challenge, the control groups showed anorexia and soft poop, whereas the experimental group showed few anorexia symptoms. Significantly different from the control group, the relative weight gain ratios of the immunized rEmMIC2 and rEmMIC3 groups were 78.37% and 75.29%, respectively, and the oocyst reduction was 77.95% and 76.09%, respectively, and the anticoccidial index was 171.12 and 169.29, respectively. IgG antibody, IFN-γ, IL-4, IL-10, and IL-17 levels were significantly increased in the experimental group. The results showed that rEmMIC2 and rEmMIC3 have potential as vaccine candidate antigens.

Immunomodulatory effects of two recombinant arginine kinases in Sarcoptes Scabiei on host peripheral blood mononuclear cells.

Background: As an important zoonotic parasitic disease with global distribution, scabies causes serious public health and economic problems. Arginine kinase (AK) is involved in cell signal transduction, inflammation, and apoptosis. Two AKs were identified in Sarcoptes scabiei, but their functions in the host immune response remain unclear. Methods: rSsAK-1 and rSsAK-2 were expressed, purified, and immunolocalized. The effects of rSsAK-1 and rSsAK-2 on rabbit PBMC proliferation, apoptosis, and migration; Bcl-2, Bcl-xl, Fas, Bax, and NF-κB transcription levels; and IL-2, IFN-γ, IL-4, IL-10, TGF-ß1, and IL-17 secretion were detected. Results: rSsAK-1 and rSsAK-2 were cloned and expressed successfully. Both enzymes were ~57 kDa and contained 17-kDa tagged proteins, and had good catalytic activity and immunoreactivity. The proteins were located in the S. scabiei exoskeleton, chewing mouthparts, legs, stomach, and intestine. SsAK-1 and SsAK-2 were secreted in the pool and epidermis of the skin lesions, which may be involved in S. scabiei-host interaction. rSsAK-1 and rSsAK-2 significantly promoted cell proliferation, induced cell migration, inhibited apoptosis, and increased Bcl-2, Bcl-xl and NF-κB (p65) transcription levels concentration-dependently, and inhibited IL-2, IFN-γ, and IL-10 secretion and promoted IL-4 and IL-17 secretion. Conclusion: rSsAK-1 and rSsAK-2 might increase Bcl-2 and Bcl-xl expression by activating the NF-κB signaling pathway to promote cell proliferation and inhibit apoptosis, which induced PBMC survival. By inducing PBMC migration to the infection site, rSsAK-1 and rSsAK-2 shifted the Th1/Th2 balance toward Th2 and changed the Th17/Treg balance, which indicated their immune role in S. scabiei allergic inflammation.

Preliminary evaluation of the protective effects of recombinant AMA1 and IMP1 against Eimeria stiedae infection in rabbits.

BACKGROUND: Eimeria stiedae parasitizes the bile duct, causing hepatic coccidiosis in rabbits. Coccidiosis control using anticoccidials led to drug resistance and residues; therefore, vaccines are required as an alternative control strategy. Apical membrane antigen 1 (AMA1) and immune mapped protein 1 (IMP1) are surface-located proteins that might contribute to host cell invasion, having potential as candidate vaccine antigens. METHODS: Herein, we cloned and expressed the E. stiedae EsAMA1 and EsIMP1 genes. The reactogenicity of recombinant AMA1 (rEsAMA1) and IMP1 (rEsIMP1) proteins were investigated using immunoblotting. For the vaccination-infection trial, rabbits were vaccinated with rEsAMA1 and rEsIMP1 (both 100 µg/rabbit) twice at 2-week intervals. After vaccination, various serum cytokines were measured. The protective effects of rEsAMA1 and rEsIMP1 against E. stiedae infection were assessed using several indicators. Sera were collected weekly to detect the specific antibody levels. RESULTS: Both rEsAMA1 and rEsIMP1 showed strong reactogenicity. Rabbits vaccinated with rEsAMA1 and rEsIMP1 displayed significantly increased serum IL-2 (F (4, 25) = 9.53, P = 0.000), IL-4 (F (4, 25) = 7.81, P = 0.000), IL-17 (F (4, 25) = 8.55, P = 0.000), and IFN-γ (F (4, 25) = 6.89, P = 0.001) levels; in the rEsIMP1 group, serum TGF-ß1 level was also elevated (F (4, 25) = 3.01, P = 0.037). After vaccination, the specific antibody levels increased and were maintained at a high level. The vaccination-infection trial showed that compared with the positive control groups, rabbits vaccinated with the recombinant proteins showed significantly reduced oocyst output (F (5, 54) = 187.87, P = 0.000), liver index (F (5, 54) = 37.52, P = 0.000), and feed conversion ratio; body weight gain was significantly improved (F (5, 54) = 28.82, P = 0.000). CONCLUSIONS: rEsAMA1 and rEsIMP1 could induce cellular and humoral immunity, protecting against E. stiedae infection. Thus, rEsAMA1 and rEsIMP1 are potential vaccine candidates against E. stiedae.

Effects of Sarcoptes scabiei Translationally Controlled Tumor Protein (TCTP) on Histamine Release and Degranulation of KU812 Cells.

Scabies is a common parasitic dermatological infection worldwide that is often neglected. Scabies mites stimulate host inflammatory symptoms via secreted and excreted proteins, which induce basophil and mast cell degranulation and host histamine release. However, the mechanism of degranulation and histamine release is unclear. Moreover, the Sarcoptes scabiei translationally controlled tumor protein (TCTP) is predicted as an excreted protein, which may be involved in host inflammatory response regulation. First, we evaluated S. scabiei TCTP gene (SsTCTP) transcription in larvae, nymphs, and adults by qRT-PCR, and SsTCTP transcription was highest in larvae, followed by nymphs. Second, we found that the S. scabiei TCTP recombinant protein (rSsTCTP) promoted mice histamine release in vivo by Evans blue Miles assay. Therefore, to further explore the possible role of S. scabiei TCTP in host inflammatory response regulation, we established a degranulation model of KU812 cells. The results of the degranulation model suggested that rSsTCTP could induce enhanced degranulation of KU812 cells and increase the secretion of histamine and the expression of IL-4, IL-6, and IL-13 in vitro. In conclusion, we speculate that scabies mites could stimulate host histamine release and Th2 response by excreting S. scabiei TCTP.

Comprehensive Molecular Characterization of the Mitochondrial Genome of the Takin Lungworm Varestrongylus eleguneniensis (Strongylida: Protostrongylidae).

The takin lungworm Varestrongylus eleguneniensis (Strongylida: Protostrongylidae) causes lethal bronchopneumonia and represents severe threats to captive and wild populations. However, until now there has been very limited information available concerning the molecular epidemiology and evolutionary biology of V. eleguneniensis. Mitochondrial genomes (mtDNAs) can provide resources for investigations in these areas and, therefore, can assist with the surveillance and control of this lungworm. Herein, the complete mtDNA of V. eleguneniensis was sequenced and characterized with Illumina pipeline analyses. This circular genome (13,625 bp) encoded twelve protein-coding genes (PCGs), two rRNAs, and twenty-two tRNAs, with notable levels of AT and GC skews. Comparative genomics revealed a purifying selection among PCGs, with cox1 and nad6 having the lowest and the highest evolutionary rate, respectively. Genome-wide phylogenies showed a close relationship between V. eleguneniensis and Protostrongylus rufescens in Strongylida. Single gene (PCGs or rRNAs)-based phylogenies indicated that cox1 and nad5 genes shared the same family-level topology with that inferred from genomic datasets, suggesting that both genes could be suitable genetic markers for evolutionary and phylogenetic studies of Strongylida species. This was the first mtDNA of any member of the genus Varestrongylus, and its comprehensive molecular characterization represents a new resource for systematic, population genetic and evolutionary biological studies of Varestrongylus lungworms in wildlife.

Comparative genomics of Sarcoptes scabiei provide new insights into adaptation to permanent parasitism and within-host species divergence.

Sarcoptes scabiei is the causative agent of a highly contagious skin disease in humans and more than 100 mammals. Here, we reported the first chromosome-level reference genome of S. scabiei isolated from rabbits, with a contig N50 size of 5.92 Mb, a total assembled length of 57.30 Mb, ∼12.65% repetitive sequences and 9333 predicted protein-coding genes. The phylogenetic tree based on 1338 shared high-confidence single-copy orthologous genes estimated that the mammalian ectoparasite S. scabiei and the plant-feeding mite Tetranychus urticae separated ∼340 million years ago. Both neighbour-joining tree and principal component analysis of 20 mite populations isolated from four hosts (humans, pigs, dogs and rabbits) distributed in three countries (China, Australia and the USA) consistently supported genetic subdivisions according to host species rather than geographical location. The demographic history of S. scabiei reconstructed by multiple sequentially Markovian coalescent analysis suggested that S. scabiei isolated from rabbits, humans, dogs and pigs diverged ∼5000 years ago. Investigation of the homeobox (Hox) genes revealed that S. scabiei contains 8 of 10 canonical Hox genes that are present in the arthropod ancestor, and the absence of the Abd-A gene may correlate with the long gap between their front and back legs. Comparative genomics demonstrated that genes specific to scabies mites were mainly enriched in nutrition digestive systems, whereas genes in the families that involved detoxification (cytochrome P450, carboxyl/cholinesterases and the ATP-binding cassette transporter C group) were extremely contracted compared with that of other mites analysed in this study. Selective sweep analysis of mite populations from various pairs of two out of the four host species revealed that the strongest selective sweep signals were mainly enriched in cysteine-type peptidase activity and apoptosis. The results provided clues for the mechanisms of S. scabiei adaptation to a permanent parasitic lifestyle and knowledge that would enable further control of this highly contagious skin disease.

Prokaryotic Expression of Eimeria magna SAG10 and SAG11 Genes and the Preliminary Evaluation of the Effect of the Recombinant Protein on Immune Protection in Rabbits.

Eimeria magna is a common coccidia in the intestines of rabbits, causing anorexia, weight loss, diarrhea, and bloody stools. This study cloned and determined the expression levels of four Eimeria surface antigens (EmSAGs) at different developmental stages and showed that EmSAG10 and EmSAG11 are highly expressed at the merozoite stage. Rabbits were immunized with rEmSAG10 and rEmSAG11, and then challenged with E. magna after 2 weeks. Serum-specific antibodies and cytokine levels were detected using ELISA. Immune protection was evaluated based on the rate of the oocysts decrease, the output of oocysts (p < 0.05), the average weight gain, and the feed: meat ratio. Our results showed that rabbits immunized with rEmSAG10 and rEmSAG11 had a higher average weight gain (62.7%, 61.1%), feed; meat ratio (3.8:1, 4.5:1), and the oocysts decrease rate (70.8%, 81.2%) than those in the control group, and also significantly reduced intestinal lesions. The specific IgG level increased one week after the first rEmSAG10 and rEmSAG11 immunization and was maintained until two weeks after the challenge (p < 0.05). The TGF-ß, IL-4, and IL-10 levels in the serum increased significantly after the secondary immunization with rEmSAG10 and rEmSAG11, while the IL-2 levels increased significantly after the secondary immunization with rEmSAG11 (both p < 0.05), suggesting that rEmSAG10 can induce a humoral and cellular immunity, while rEmSAG11 can only induce a humoral immunity. Therefore, rEmSAG10 is a candidate antigen for E. magna recombinant subunit vaccines.

Characterization of a novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae and its role in regulating mice immune cell response.

Baylisascaris schroederi (B. schroederi) is a severe threat to the survival of giant pandas. Currently, the immune regulation mechanism of B. schroederi is poorly understood. Cysteine protease inhibitors (CPI) play important roles in the regulation of host immune responses against certain nematodes. In this study, a recombinant CPI of B. schroederi migratory larvae (rBsCPI-1) was cloned and expressed, and the effects of rBsCPI-1 on the physiological activities and antigen presentation of monocyte-derived macrophages (MDMs) were analyzed. We also analyzed the regulatory effects of rBsCPI-1 on the proliferation and differentiation of CD4+ T cells. And further identified the signaling pathways which play important roles in this process. The results showed that rBsCPI-1 activated the TLR2/4-small Rho GTPases-PAK1 pathway. On the one hand, it increased the phagocytosis and migration of MDMs. On the other hand, it activated downstream MAPK and NF-κB signaling pathways to induce apoptosis of MDMs. rBsCPI-1 also induced MDMs to polarize to the M2 subtype, thereby exerting an immunosuppressive effect. Meanwhile, rBsCPI-1 inhibited the antigen presentation process by decreasing the expression of MHC-II molecules, further inhibiting the proliferation of CD4+ T cells and inducing a Th1/Th2 mixed immune response. Treg cells with immunosuppressive effects were increased. The PD-L2/PD-1 and CD80/CTLA-4 signaling pathways between MDMs and CD4+ T cells were also activated by rBsCPI-1. In conclusion, this study preliminarily confirmed that rBsCPI-1 affects the physiological activities and polarization of MDMs through the TLR2/4 signaling pathway, and further interferes with antigen presentation response, inducing CD4+ T cells to play an immunosuppressive cellular response during the migratory process of B. schroederi. Thus, this study will provide a reference for elucidating the immune evasion mechanism of B. schroederi and developing new drugs and protective vaccines against B. schroederi.

A novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae regulates inflammasome activation through the TLR4-ROS-NLRP3 pathway.

BACKGROUND: Giant pandas (Ailuropoda melanoleuca) are the obligate host of the parasitic roundworm Baylisascaris schroederi. The infection of giant pandas with B. schroederi is very common. At present, little is known about the mechanism of immune interaction between B. schroederi and the host. As an important component of innate immunity, the NOD-like receptor 3 (NLRP3) inflammasome plays an important role in host immune response and the occurrence and development of infectious diseases. METHODS: We analyzed the regulation of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) by the recombinant B. schroederi migratory larvae cysteine protease inhibitor rBsCPI-1, knowing from a previous study that the CPI-1 is highly expressed in B. schroederi migratory larvae. We first determined the effects of rBsCPI-1 and excretory-secretory products of B. schroederi migratory larvae on cell proliferation using the CCK-8 and LDH release assays. We then analyzed NLRP3 inflammasome activation, pyroptosis and pro-inflammatory cytokine release by quantitative-PCR, western blotting and enzyme-linked immunosorbent assay. The signaling pathway of rBsCPI-1 to activate NLRP3 inflammasomes was analyzed in activation and inhibition experiments. Finally, the effects of rBsCPI-1 on inflammasome activation in mice immunized with rBsCPI-1 were analyzed. RESULTS: The activation and inhibition experiments revealed that rBsCPI-1 induced inflammasome activation through the TLR4-ROS-NLRP3 signaling pathway, with reactive oxygen species (ROS) not only functioning as an activator of the NLRP3 inflammasome, but also an activation product of the NLRP3 inflammasome. rBsCPI-1 promoted the activation and assembly of the NLRP3 inflammasome, which further converted the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 into mature active forms. At the same time, caspase-1 cleaved gasdermin D to trigger cell pyroptosis. The results of animal immunization experiments further confirmed that rBsCPI-1 could induce the activation of the NLRP3 inflammasome. CONCLUSIONS: rBsCPI-1 activates the inflammasome through the TLR4-ROS-NLRP3 signaling pathway and further induces the pyroptosis of MDMs and release of pro-inflammatory factors IL-1ß and IL-18, thus promoting the occurrence and development of the inflammatory response in the host.

Regulatory effects of a novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae on mice immune cells.

BACKGROUND: The giant panda (Ailuropoda melanoleuca) is a well-known, rare and endangered species. Baylisascaris schroederi is a pathogenic ascarid. Infection with B. schroederi may cause death in giant pandas. At present, the immune evasion mechanism of B. schroederi is little known. Cysteine protease inhibitors (CPI) play important roles in the regulation of host immune responses against certain nematodes. In this study, we focused on the analysis of the regulation of B. schroederi migratory larvae CPI (rBsCPI-1) on mice immune cells. METHODS: First, the pattern recognition receptors on the surface of peripheral blood mononuclear cells (PBMCs) and the signal pathways that transduce extracellular signals into the nucleus activated by rBsCPI-1 were identified. Then, the regulatory effects of rBsCPI-1 on PBMCs physiological activities were detected. Finally, the effects of rBsCPI-1 on TLR signaling pathway activation and NF-κB phosphorylation in mice immunized with recombinant protein were analysed. RESULTS: The results suggested that rBsCPI-1 secreted by B. schroederi migratory larvae is mainly recognized by TLR2 and TLR4 on PBMCs. Extracellular signals are transduced into the nucleus through the MAPK and NF-κB signaling pathways, enhancing the phagocytosis, migration, and apoptosis of PBMCs; meanwhile, rBsCPI-1 induces high expression of NO. Thus, rBsCPI-1 plays a role in immune regulation. In addition, the high expression of negative regulatory factors also ensured that TLR activation is maintained at the optimal level. CONCLUSIONS: rBsCPI-1 can transduce regulatory signals into immune cells by activating the TLR2/4-NF-κB/MAPK signaling pathway, having a certain regulatory effect on the physiological activities. Meanwhile, rBsCPI-1 can maintain the immune response in a balance by limiting the over-activation of the TLRs signaling pathway and thus contributes to B. schroederi immune evasion.

Transcriptome Analysis of Otodectes cynotis in Different Developmental Stages.

The mite Otodectes cynotis is distributed worldwide and parasitism the ear canals of cats and dogs, causing otitis externa. Molecular biology of O. cynotis is poorly understood, with only a few genes being deposited in public databases. In the present study, we aimed to perform transcriptome analysis of O. cynotis using SMRT and Illumina sequencing of RNA from different development stages. SMRT-Seq of O. cynotis demonstrated 5,431 final transcripts, including 406 long non-coding RNAs and 2,698 differentially expressed genes (DEGs), including 1,357 up-regulated genes and 1,341 down-regulated genes between adult mites and nymph/larva. A total of 397 putative allergen genes were detected, 231 of which were DEGs. Among them, 77 were homologous of known mite allergens. The expression level of allergen genes hints at the pathogenicity of mites in different life stages, and the protein interaction network analysis could identify possible key genes in the pathogenic mechanism. Intriguingly, Gene Ontology analysis showed that most of the (DEGs) were associated with the terms hydrolase activity and proteolysis. Kyoto Encyclopedia of genes and genomes (KEGG) analysis identified drug metabolism-cytochrome P450 signal pathway as one of the top pathways. SMRT-Seq of the full-length transcriptome of O. cynotis was performed first, and a valuable resource was acquired through the combination analysis with the Illumina sequencing data. The results of our analyses provide new information for further research into Otodectes cynotis.

Genetic diversity of Echinococcus granulosus sensu lato in China: Epidemiological studies and systematic review.

Cystic echinococcosis (CE) is a neglected tropical zoonosis caused by Echinococcus granulosus sensu lato (s.l.) and remains a major public health concern globally. Here, CE isolates (n = 3310) with clearly defined genotypes and geographical origins in China were retrieved through our epidemiological survey (2016-2020) and systematic review (1992-2020). Existing known genotypes of Echinococcus granulosus sensu lato (E. granulosus s.l.) except for G4 have been found in China, particularly on the Tibetan Plateau, where their genetic diversity is unique to that part of the world. According to the systematic review, genetic compositions of E. granulosus s.l. in China were as follows: E. granulosus (G1, G3), 98.3%; Echinococcus ortleppi (G5), 0.1%; Echinococcus intermedius (G6, G7), 1.4%; and Echinococcus canadensis (G8, G10), 0.2%. Specifically, G1 was responsible for 97.7% of infections and characterized by the broadest host ranges and geographic distributions. Our epidemiological results showed a relatively stable genetic composition of E. granulosus s.l. in sheep and yaks from three CE hyperendemic provinces (Xinjiang, Sichuan, Qinghai). A higher proportion of fertile cysts were found in sheep (287/406, 70.7%) than in yaks (28/184, 15.2%). During the past 29 years, 51 cox1 haplotypes of E. granulosus s.l. were endemic in China. The ancestral haplotype (Hap_2) remained the most common haplotype, 12 relatively common haplotypes were endemic and nine newly reported haplotypes were found during the survey. Overall, our results demonstrate that the compulsory immunization of sheep and the pilot EG95 vaccination campaign in yaks are well matched with the current genotypic situation. In addition to yaks, we advocate for more surveillance of CE isolates from pigs, cattle, goats and camels, since their roles in the transmission and reservation of E. granulosus s.l. have been largely ignored in China.

How does Chinese central environmental inspection affect corporate green innovation? The moderating effect of bargaining intentions.

Central environmental inspection (CEI) is an environmental governance tool launched by the Chinese central government in recent years. The aim of CEI is to enhance the implementation of environmental regulations by going beyond supervising enterprises to supervise local governments. Previous studies have shown that CEI is beneficial for improving corporate environmental performance, but the relationship between CEI and the environmental behavior of companies achieving a high level of environmental performance is not clear. Drawing on principal-agent theory, this study explains how CEI influences corporate green innovation by changing the incentive structures of local governments and firms for adopting different environmental strategies. Moreover, two boundary conditions between CEI and corporate green innovation are identified: political connections and industry competition. The boundary conditions affect the individual and collective bargaining intentions of companies to comply with environmental regulations. We test our hypotheses by using a sample of 1152 publicly traded industrial firms in China from 2014 to 2018. Our findings show that CEI is conducive to corporate green innovation. This positive effect is strengthened in politically connected firms or firms in highly monopolized industries with lower bargaining intentions. Our study contributes to a better understanding of the effect of CEI on micro (firm-level) environmental behaviors and the antecedents of corporate green innovation.

Genome of the Giant Panda Roundworm Illuminates Its Host Shift and Parasitic Adaptation.

Baylisascaris schroederi, a roundworm (ascaridoid) parasite specific to the bamboo-feeding giant panda (Ailuropoda melanoleuca), represents a leading cause of mortality in wild giant panda populations. Here, we present a 293-megabase chromosome-level genome assembly of B. schroederi to infer its biology, including host adaptations. Comparative genomics revealed an evolutionary trajectory accompanied by host-shift events in ascaridoid parasite lineages after host separations, suggesting their potential for transmission and rapid adaptation to new hosts. Genomic and anatomical lines of evidence, including expansion and positive selection of genes related to the cuticle and basal metabolisms, indicate that B. schroederi undergoes specific adaptations to survive in the sharp-edged bamboo-enriched gut of giant pandas by structurally increasing its cuticle thickness and efficiently utilizing host nutrients through gut parasitism. Additionally, we characterized the secretome of B. schroederi and predicted potential drug and vaccine targets for new control strategies. Overall, this genome resource provides new insights into the host adaptation of B. schroederi to the giant panda as well as the host-shift events in ascaridoid parasite lineages. Our findings on the unique biology of B. schroederi will also aid in the development of prevention and treatment measures to protect giant panda populations from roundworm parasitism.