Effects of radiation on the fitness, sterility and arbovirus susceptibility of a Wolbachia-free Aedes albopictus strain for use in the sterile insect technique.

BACKGROUND: The sterile insect technique (SIT) is a green and species-specific insect pest control technique that suppresses target populations by releasing factory-reared, radiosterilized males into the wild. Once released, it is important to be able to distinguish the released males from the wild males for monitoring purposes. Several methods to mark the sterile males exist. However, most have limitations due to monetary, process efficiency, or insect quality. Aedes albopictus is naturally infected with Wolbachia at a high prevalence, therefore the elimination of Wolbachia can serve as a biomarker to distinguish factory-reared male mosquitoes from wild conspecifics. RESULTS: In this study, a Wolbachia-free Ae. albopictus GT strain was developed and its fitness evaluated, which was found to be comparable to the wild GUA strain. In addition, GT male mosquitoes were irradiated at the adult stage and a dose of 20 Gy or more induced over 99% sterility. Moreover, a dose of 30 Gy (almost completely sterilizing male and female mosquitoes) had limited effects on the mating competitiveness of GT males and the vector competence of GT females, respectively. However, radiation reduced mosquito longevity, regardless of sex. CONCLUSION: Our results indicate that the Ae. albopictus GT strain can be distinguished from wild mosquitoes based on Wolbachia status and shows similar fitness, radio-sensitivity and arbovirus susceptibility to the GUA strain, indicating that it is feasible to use the GT strain to suppress Ae. albopictus populations for SIT programmes. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

iNOS is essential to maintain a protective Th1/Th2 response and the production of cytokines/chemokines against Schistosoma japonicum infection in rats.

Humans and a wide range of mammals are generally susceptible to Schistosoma infection, while some rodents such as Rattus rats and Microtus spp are not. We previously demonstrated that inherent high expression levels of nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), plays an important role in blocking the growth and development of Schistosoma japonicum in wild-type rats. However, the potential regulatory effects of NO on the immune system and immune response to S. japonicum infection in rats are still unknown. In this study, we used iNOS-knockout (KO) rats to determine the role of iNOS-derived NO in the immune system and immunopathological responses to S. japonicum infection in rats. Our data showed that iNOS deficiency led to weakened immune activity against S. japonicum infection. This was characterized by the impaired T cell responses and a significant decrease in S. japonicum-elicited Th2/Th1 responses and cytokine and chemokine-producing capability in the infected iNOS-KO rats. Unlike iNOS-KO mice, Th1-associated cytokines were also decreased in the absence of iNOS in rats. In addition, a profile of pro-inflammatory and pro-fibrogenic cytokines was detected in serum associated with iNOS deficiency. The alterations in immune responses and cytokine patterns were correlated with a slower clearance of parasites, exacerbated granuloma formation, and fibrosis following S. japonicum infection in iNOS-KO rats. Furthermore, we have provided direct evidence that high levels of NO in rats can promote the development of pulmonary fibrosis induced by egg antigens of S. japonicum, but not inflammation, which was negatively correlated with the expression of TGF-ß3. These studies are the first description of the immunological and pathological profiles in iNOS-KO rats infected with S. japonicum and demonstrate key differences between the responses found in mice. Our results significantly enhance our understanding of the immunoregulatory effects of NO on defensive and immunopathological responses in rats and the broader nature of resistance to pathogens such as S. japonicum.

Immunopathology in schistosomiasis is regulated by TLR2,4- and IFN-γ-activated MSC through modulating Th1/Th2 responses.

BACKGROUND AND AIMS: A marked egg-induced CD4+ T cell programmed inflammation and subsequent hepatic fibrosis characterize the pathogenesis of schistosomiasis. Mesenchymal stem cell (MSC) has been extensively studied for the treatment of schistosomiasis. However, the mechanism by which MSCs modulate the pathogenesis of schistosomiasis has not been clarified. Furthermore, the local inflammatory milieu may greatly influence the immunoregulatory properties of MSCs, and our early experiments demonstrated that Toll-like receptor (TLR)2/TLR4 agonist effected immune modulation of MSC. Here, we further investigated their modulation on the pathogenesis of schistosomiasis. METHODS: Adult BALB/c male mice were percutaneously infected with 16 ± 2 pairs S. japonicum cercariae and received intravenously pretreated MSC at 1 week and 3 weeks post-infection, respectively. At 8 weeks post-infection, effects of MSC on liver histology were shown by hematoxylin and eosin (H&E) staining and Masson staining and quantitatively compared by the hepatic hydroxyproline content; α-smooth muscle actin (α-SMA), collagen type I(Col-1), transforming growth factor ß (TGF-ß), and tumor necrosis factor-α (TNF-α) gene expression in the liver were assessed by semi-quantitative polymerase chain reaction (PCR); the Th1/Th2 dominance among different groups was compared by analyzing CD4+ interferon-γ (IFN-γ)+ and CD4+interleukin-4 (IL-4)+T cells in the liver by flow cytometry and serum level of IFN-γ and IL-5 using enzyme-linked immunosorbent assay (ELISA). Effects of different kinds of MSC were further evaluated in vitro by the coculture system. RESULTS: Results showed TLR4- and IFN-γ-activated MSC alleviated liver fibrosis in infected mice, without a significant increase of mortality, and unpretreated MSC showed no clear improvement; however, TLR2- and IFN-γ-activated MSC displayed aggravated immunopathology. In accord with the pathological results, TLR4- and IFN-γ-activated MSC groups showed moderate enhancement of Th1 response in vitro and clear Th1 dominance in vivo without leading to extreme inflammation, whereas TLR2- and IFN-γ-activated MSC not only induced Th1 response, but also triggered excessive inflammation as evidenced by atrophy of the thymus and higher TNF level in the coculture system. CONCLUSIONS: This study demonstrates that TLR4 combined with IFN-γ can activate the MSC group with positive effects on the pathology of schistosomiasis by modulating Th subsets at some degree. This result suggests that when MSC is being used to treat different immuno-disturbance complications, subtle pretreatment methods should be seriously considered.

IL-17A neutralizing antibody attenuates eosinophilic meningitis caused by Angiostrongylus cantonensis by involving IL-17RA/Traf6/NF-κB signaling.

BACKGROUND: Angiostrongylus cantonensis (A. cantonensis) is a foodborne parasite that can invade the central nervous system (CNS), resulting in eosinophilic meningitis (EM). However, the mechanism by which A. cantonensis causes eosinophilic infiltration into CNS is not well understood. METHODS: In this study eosinophilic infiltration into the CNS caused by A. cantonensis was assessed based on eosinophil counts and evaluation of interleukin (IL)-5 and -13 levels by real-time PCR in brain of Balb/c mice. The expression and activation of IL-17A, IL17 receptor (IL-17R A), and IL-17RC and the related signaling molecules nuclear factor (NF)-κB1, NF-κB2, NF-κB activator (Act)1, tumor necrosis factor receptor-associated factor (Traf)5, and Traf6 during A. cantonensis infection in brain tissue of Balb/c mice were examined by real-time, western blotting and immunofluroence. A. cantonensis-infected Balb/c mice were treated with IL-17A neutralizing antibody to evaluate the role of IL17A in eosinophil accumulation in the CNS. RESULTS: Our results showed A. cantonensis infection caused eosinophil accumulation and alterations in IL-5 and -13 levels. The expression of IL-17A and -17RA, Act1, and Traf6 but not of IL-17RC and Traf5 was upregulated during infection; this was accompanied by NF-κB1 and -κB2 activation. Importantly, application of IL-17A neutralizing antibody attenuated eosinophil accumulation in CNS and reversed the changes in IL-5 and -13 expression caused by A. cantonensis infection. Additionally, IL-17RA and Traf6 levels decreased, which was accompanied by NF-κB inactivation. CONCLUSION: IL-17A plays an important role in EM caused by A. cantonensis, possibly through activation of NF-κB via the IL-17RA/Traf6 signaling pathway. These findings highlight the potential for using IL-17A neutralizing antibody as a therapeutic strategy for the treatment of EM.

Snail-borne parasitic diseases: an update on global epidemiological distribution, transmission interruption and control methods.

BACKGROUND: Snail-borne parasitic diseases, such as angiostrongyliasis, clonorchiasis, fascioliasis, fasciolopsiasis, opisthorchiasis, paragonimiasis and schistosomiasis, pose risks to human health and cause major socioeconomic problems in many tropical and sub-tropical countries. In this review we summarize the core roles of snails in the life cycles of the parasites they host, their clinical manifestations and disease distributions, as well as snail control methods. MAIN BODY: Snails have four roles in the life cycles of the parasites they host: as an intermediate host infected by the first-stage larvae, as the only intermediate host infected by miracidia, as the first intermediate host that ingests the parasite eggs are ingested, and as the first intermediate host penetrated by miracidia with or without the second intermediate host being an aquatic animal. Snail-borne parasitic diseases target many organs, such as the lungs, liver, biliary tract, intestines, brain and kidneys, leading to overactive immune responses, cancers, organ failure, infertility and even death. Developing countries in Africa, Asia and Latin America have the highest incidences of these diseases, while some endemic parasites have developed into worldwide epidemics through the global spread of snails. Physical, chemical and biological methods have been introduced to control the host snail populations to prevent disease. CONCLUSIONS: In this review, we summarize the roles of snails in the life cycles of the parasites they host, the worldwide distribution of parasite-transmitting snails, the epidemiology and pathogenesis of snail-transmitted parasitic diseases, and the existing snail control measures, which will contribute to further understanding the snail-parasite relationship and new strategies for controlling snail-borne parasitic diseases.

Infection by the nematode Angiostrongylus cantonensis induces differential expression of miRNAs in mouse brain.

BACKGROUND: The parasitic nematode Angiostrongylus cantonensis is the primary pathogen causing eosinophilic meningitis and meningoencephalitis in nonpermissive hosts. The larval parasites are eliminated by the host's immune responses in the central nervous system (CNS) through infiltration of eosinophils and lymphocytes. This study aimed to determine primary alterations of microRNA (miRNA) during A. cantonensis infection in mice. METHODS: miRNA array was used to analyze the expression of miRNA in uninfected and A. cantonensis-infected mouse brains at 21 days postinfection (dpi). Target genes were predicted by miRDB software, and protein-protein interaction network was analyzed using STRING v9.1. Expression levels of selected miRNAs and cytokine production were verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Twenty-five mature miRNAs showed differential expression in infected mouse brains, of which 24 were upregulated and one was downregulated compared to the uninfected control. These 25 miRNAs were divided into five clusters, and the first upregulated cluster was selected for further bioinformatics analysis. Target gene prediction and gene ontology (GO) enrichment analysis revealed that the miRNAs were mainly related to the immune response. Furthermore, six target genes of mmu-miR-146a-5p were predicted to interact with tumor necrosis factor alpha (TNF-α). The in vitro study suggested that transfected mmu-miR-146a-5p inhibitor upregulated TNF-α and its target gene Traf6 in microglia following stimulation with A. cantonensis larval antigen. CONCLUSION: This study suggested a critical role of miRNAs in the host defense during A. cantonensis infection, providing new insights into the molecular mechanisms underlying the interaction between mmu-miR-146a-5p and TNF-α in angiostrongyliasis in nonpermissive hosts.

Parasitology should not be abandoned: data from outpatient parasitological testing in Guangdong, China.

Over the past six decades, the Chinese government made parasitoses with a high disease burden, including soil-transmitted nematode infections, malaria, leishmaniasis, filariasis, and schistosomiasis, a public health priority because they were seen to be crucial impediments to the development of rural areas. As a result, these debilitating parasitic diseases that used to be widely prevalent have been well controlled or eliminated. Consequently, less attention has been paid to parasitic infection during the rapid development of the economy, especially in developed areas. However, our investigations conducted in the parasitological laboratory of Sun Yat-sen University (Guangzhou, Guangdong, China) show that emerging parasitic diseases still threaten many people's health, with 340 of 880 outpatients (38.6%) receiving a diagnosis of parasitic disease, among whom 201 (59.1%) had clonorchiasis and 120 (35.3%) had taeniasis/cysticercosis. Furthermore, our doctors are not equipped with sufficient parasitology knowledge because this discipline is not able to maintain attraction. Many parasitic infections that result in severe consequences are treatable and preventable, but the phenomena of misdiagnosis and missed diagnosis are common and merit attention.

Nitric oxide blocks the development of the human parasite Schistosoma japonicum.

Human schistosomiasis, caused by Schistosoma species, is a major public health problem affecting more than 700 million people in 78 countries, with over 40 mammalian host reservoir species complicating the transmission ecosystem. The primary cause of morbidity is considered to be granulomas induced by fertilized eggs of schistosomes in the liver and intestines. Some host species, like rats (Rattus norvegicus), are naturally intolerant to Schistosoma japonicum infection, and do not produce granulomas or pose a threat to transmission, while others, like mice and hamsters, are highly susceptible. The reasons behind these differences are still a mystery. Using inducible nitric oxide synthase knockout (iNOS-/-) Sprague-Dawley rats, we found that inherent high expression levels of iNOS in wild-type (WT) rats play an important role in blocking growth, reproductive organ formation, and egg development in S. japonicum, resulting in production of nonfertilized eggs. Granuloma formation, induced by fertilized eggs in the liver, was considerably exacerbated in the iNOS-/- rats compared with the WT rats. This inhibition by nitric oxide acts by affecting mitochondrial respiration and energy production in the parasite. Our work not only elucidates the innate mechanism that blocks the development and production of fertilized eggs in S. japonicum but also offers insights into a better understanding of host-parasite interactions and drug development strategies against schistosomiasis.

A case report: A rare case of infant gastrointestinal canthariasis caused by larvae of Lasioderma serricorne (Fabricius, 1792) (Coleoptera: Anobiidae).

BACKGROUND: Canthariasis is a disease of humans caused by the infestation of beetle larvae. It is the second important insectal disease after myiasis. Several species of beetles are reported to cause the disease in gastrointestinal tract, urogenital system, nasal sinuses, ears and faces of mammals. The cigarette beetle Lasioderma serricorne is a widespread and destructive pest that usually feeds on tobacco, tea, beans, cereal grains, and animal and plant specimen. While there was no previous evidence of human infestation by this worm, we report the first case of L. serricorne infestation in a baby girl in China. CASE PRESENTATION: Here the case, an eight-month-old baby girl with irritable feeling, rubbing eyes, history of contact with mud and eating oranges twice during five days before attendance, and having "worms" in her stool was admitted to the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. The clinical examination revealed that the pulse rate, blood pressure and temperature were regular, and the examination of the head, neck, and chest were unremarkable. The stool specimens containing "worms" were sent to the Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University. The worms were recovered, studied morphologically using naked eyes and anatomical lens, PCR analyzed targeting cytochrome oxidase subunit 1 (COX1) and 18S rRNA genes, examined by sequence analyses of the PCR products and finally classified by phylogenetic analysis to identify their species. Based on the findings, the worms were diagnosed as the larvae of L. serricorne. CONCLUSION: This report implies that the baby had an infestation with the larvae of L. serricorne in the gastrointestine. During contact with mud or eating oranges by the girl, worm eggs were swallowed into the stomach and resisted gastric acid digestion which eventually hatched into larvae and caused canthariasis. The 8 months girl had underdeveloped immune system which might facilitate the disease. This report implicates that L. serricorne can infest human accidentally and cause canthariasis that may lead to severe damage to infant and older patient upon involvement of important organs of the body. The patients once diagnosed having canthariasis should be treated in time.

[Pathology and pathogenesis of liver fibrosis induced by Schistosoma japonicum].

Schistosomiasis is a widely distributed parasitic zoonoses that threatens human' s health and social economic development. China is one of the most endemic countries in the world. Schistosoma egg are mainly lodged in the liver and intestinal tissues. There, the eggs induce a granulomatous host immune response largely characterized by lymphocytes, eosinophils, and alternatively activated macrophages. The process of granuloma formation induces chronic inflammation that leads to liver fibrosis accompanied by obvious manifestations such as hepatosplenomegaly and ascites. In this article, we review the advanced progress in research about schistosomiasis hepatic fibrosis, including the pathology of liver fibrosis, the formation and modulation of granuloma and fibrosis, the key inflammatory factors and related signaling pathways, the regulatory role of ncRNA in the process of fibrosis, and anti-fibrosis treatment and new drugs development.

The expression of molecule CD28 and CD38 on CD4⁺/CD8⁺ T lymphocytes in thymus and spleen elicited by Schistosoma japonicum infection in mice model.

Schistosomiasis caused by human schistosomes such as Schistosoma japonicum (S. japonicum) is considered as an immune-related disease. It was demonstrated that specific cytokine antibodies' response elicited by S. japonicum infection was gradually downregulated with the progress of the disease, resulting in a Th1/Th2 polarization and suppression of immune response. CD28 (cluster of differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival, and CD38 is an activating marker of T lymphocyte with high expression in many acute or chronic infections. The immune signature of CD28null T cells in the peripheral circulation associates with chronic inflammation in many diseases, such as HIV and CMV infection. In the thymus, CD28 expression on developing thymocytes appears to play a role for their selection, and it synergizes with CD38 to induce apoptosis of DP (double-positive) thymocytes. Few reports about CD28 and CD38 have been published in schistosomiasis. Here, we investigated the dynamic patterns of the expression of molecules CD28 and CD38 on CD4(+)/CD8(+) T lymphocytes of the thymus and spleen in mice model with S. japonicum infection. Our data indicated that at an early period of infection, the frequency of CD8(+)CD28(-) T cell in the spleen decreased significantly, but higher at chronic infection than that in control. However, it demonstrated an increasing trend in the thymus with the progression of infection. The frequency of CD4(+)CD28(-) T cells increased from acute infection in the thymus, while from chronic infection in the spleen. The expression of CD38 on CD8(+) T cells began to increase at 4 weeks post infection both in the thymus and spleen; its elevated expression on CD4(+) T cells emerged at 6 weeks post infection in the thymus and at 10 weeks post infection in the spleen. Praziquantel (PZQ) treatment could partially restore the frequency of CD28(+) T cell of CD4(+) T cells and CD38(+) T cell of CD8(+)/CD4(+) T cells in the spleen and CD38(+) T cell in the thymus. We hypothesized that the reactivation of S. japonicum infection may trigger expansion of CD28(-) T cells and hence mediate systemic inflammation. We speculated that CD8(+)CD28(-) T cell might be involved in immune modulation and CD8(+)CD28(-) T cell may be a crucial part in pathogenesis, which can provide further knowledge of the sophisticated mechanism of immuno-downregulation in schistosomiasis and potential treatment target.

Outer membrane vesicles isolated from two clinical Acinetobacter baumannii strains exhibit different toxicity and proteome characteristics.

Outer membrane vesicles (OMVs) are well-characterized virulence factors produced by Gram-negative bacteria. Here, we isolated two clinical Acinetobacter baumannii strains, the multidrug-resistant A. baumannii (MDRAb) A38 and non-MDRAb 5806. Strain A38 produced more abundant OMVs than strain 5806 when cultured to the early stationary phase. The results from cell proliferation assays and real-time PCR analyses indicated that A38 OMVs induced more powerful cytotoxicity and stronger innate immune responses compared with 5806 OMVs. Moreover, SDS-PAGE and LC-MS/MS analyses revealed that A38 OMVs contained more virulence factors, including Omp38, EpsA, Ptk, GroEL, hemagglutinin-like protein, and FilF. Taken together, the results of the present study suggest that MDRAb might produce abundant OMVs with more virulent factors facilitating the worse outcome, a finding that merits further study.

MicroRNA expression profile in the third- and fourth-stage larvae of Angiostrongylus cantonensis.

The pathogenesis of angiostrongyliasis, resulting from the third-stage and the fourth-stage Angiostrongylus cantonensis larvae invasion of the human central nervous system, remains elusive. MicroRNAs are important regulators of gene expression and involved in many biological processes. The aim of this study was to determine and characterize miRNAs of third (L3) and fourth (L4) larvae of A. cantonensis by Solex deep sequencing. A total of 629 conserved miRNAs (526 and 376 miRNAs in L3 and L4 larvae of A. cantonensis, respectively) and three novel candidate miRNA from L3 and L4 larva of A. cantonensis were identified with bioinformatic analysis. There were 163 miRNAs upregulated and 54 miRNAs downregulated (fold changes ≥5.0) in the L4 of A. cantonensis compared with that of L3 of A. cantonensis. Interestingly, Gene Ontology "biological process" classifications revealed that 26 miRNAs of significantly differential expression are associated with the immune system, which implies that these miRNAs might participate in the pathogenesis of angiostrongyliasis by regulating genes involved in immune response pathways. Furthermore, the differential expression patterns of 26 conserved miRNAs between L3 and L4 of A. cantonensis were verified. The results of real-time PCR and Northern blot showed that the aca-miR-124 and aca-miR-146a-5p have a low level expression in L3 larvae but high level expression in L4 larvae. Transfection of aca-miR-124 mimics alone significantly downregulated the mRNA expression of IL-6 and IL-1ß and TNF-a in the N9 cells, compared to the combination transfection of aca-miR-124 mimics and inhibitor (P < 0.05), suggesting that miR-124 of A. cantonensis have an important role in the suppression of microglia activation. In conclusion, the study presents a general picture of the expression of small RNAs in L3 and L4 of A. cantonensis and highlights conserved miRNAs differentially expressed between L3 and L4 larvae. Our data revealed that miRNAs of parasite may mediate important roles in A. cantonensis immune evasion and aca-miR-146a-5p can serve as a potential biomarker to diagnose angiostrongyliasis.

A recombined protein (rSj16) derived from Schistosoma japonicum induces cell cycle arrest and apoptosis of murine myeloid leukemia cells.

rSj16, a recombined protein from Schistosoma japonicum, has been identified as an anti-inflammatory molecule. In this study, we demonstrated that rSj16 strongly suppressed the growth of murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. rSj16 induced apoptosis by increasing the proportion of sub-G1 apoptotic cells as well as causing cell cycle arrest at the G0/G1 phase. The expressions of cyclin D1, D2, D3, and E, and Cdk 2, 4, and 6 genes in WEHI-3B JCS cells were significantly down-regulated at 24 h as measured by real-time PCR. Furthermore, apoptosis induced by rSj16 was confirmed by 4,6-diamidino-2-phenylindole nuclear staining assay and annexin V/propidium iodide double staining. A reduction of the mitochondrial membrane potential indicated an active involvement of mitochondria in the apoptosis process. rSj16 treatment induced an increase in the activity of caspase 3, 6, and 9, and expression of pro-apoptotic Bax. Meanwhile, the decreased expression of anti-apoptotic Bcl-2 was observed after rSj16 treatment. Taken together, our results implied that rSj16 can inhibit proliferation by inducing G0/G1 cell cycle arrest and apoptosis of murine myeloid leukemia cells via activation of the caspase-mediated mechanism by regulating the expression of Bcl-2 family.

The sources and metabolic dynamics of Schistosoma japonicum DNA in serum of the host.

The polymerase chain reaction (PCR) assay has turned out to be one of the most potential tools for diagnosis of schistosomiasis. However, the source and metabolic dynamics of Schistosoma japonicum DNA in the blood of hosts is not clear. In this study, rabbit models with monosexual and mixed sexual cercariae infection were established to interpret the source of the parasite DNA in serum of the hosts. Following administration of praziquantel at 7 weeks postinfection, the metabolic mechanism of S. japonicum DNA in serum of the hosts was studied. The findings showed that, for the monosexual cercariae infection, the parasite DNA was detectable in serum of the host from day 3 to week 3 postinfection, while for the mixed sexual cercariae infection, the detection results were continually positive during the 7 weeks after infection. After treatment with praziquantel, detection of S. japonicum DNA in rabbit sera became positive at the second day posttreatment, and the positive period lasted 3 weeks in the monosexual cercariae infection group. However, with the mixed sexual cercariae infection group, the PCR results remained positive for 16 weeks after treatment. We conclude that the S. japonicum DNA in host serum primarily comes from the residual body of dead schistosomula and/or tegument shedding of worm growing in the first 4 weeks postinfection, while during the spawning stage of the female schistosome, the parasite DNA mainly comes from the disintegration of inactive eggs. The duration from treatment to total elimination of worm origin DNA in serum is not exceeding 3 weeks. However, the DNA release from inactive eggs can last for more than 16 weeks. Further studies are needed to address the sources and metabolic dynamics of S. japonicum DNA in human serum.

Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis.

Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43-53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood-brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite-host interactions.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis.

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

[The immunopathology mechanisms of matrix metalloproteinases in brain infection induced by parasites].

Matrix metalloproteinase (MMP) family is a class of endogenous peptidases involved in normal physiological processes of the body, such as embryonic development, wound healing, angiogenesis, cartilage absorption, etc. However, during infection, over-expression of activated matrix metalloproteinase can lead to immunopathological change, induce disease or death, also contribute to the spread or survival of pathogens. Some human parasites such as Plasmodium, Toxoplasma gondii, Cysticercus cellulose, Angiostrongylus cantonensis larvae can invade the central nervous system, and cause inflammatory response dominated by eosinophil infiltration. This article reviews function and pathological mechanism of MMP during the infection of relevant parasites involving brain.

[The effect of gossypol, praziquantel and artemether on recombinant lactate dehydrogenase from Schistosoma japonicum].

OBJECTIVE: To detect the effect of gossypol, praziquantel and artemether on activity of the recombinant lactate dehydrogenase of Schistosoma japonicum (rSjLDH). METHODS: Effect of gossypol (0-0.10 mmol/L), praziquantel (0-0.20mmol/L) and artemether (0-0.10 mmol/L) on the enzymatic activity of rSjLDH was detected by spectrophotometric method in standard reaction system established before, same solvent of each reagent was used as control. Results were analyzed by SPSS13.0 software. RESULTS: Compared with the control, considerable inhibition for both reduction and oxidation reactions catalyzed by rSjLDH was observed at 0.04 mmol/L of gossypol (P < 0.01). The enzyme activities of rSjLDH for reduction and oxidation reactions were inhibited by 91.3% and 89.1% respectively at 0.1 mmol/L of gossypol, com-pared with the control (P < 0.01). To praziquantel, enzymatic activity inhibition was observed at 0.02 mmol/L for oxidation reaction and at 0.06 mmol/L for reduction reaction (P < 0.05). At 0.2 mmol/L of praziquantel, enzymatic activities were inhibited by 83.8% for reduction reaction and 72.2% for oxidation reaction respectively, than that of the control (P < 0.01). No inhibition for both reduction and oxidation reactions was observed at 0.1 mmol/L of artemether, compared with that of the control (P > 0.05). CONCLUSION: Gossypol and praziquantel show a high inhibition on rSjLDH. SiLDH may be one of the molecular targets of praziquantel.