Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis.

Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of ß-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes.

Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice.

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.

Modulation of the Immune Response by Nematode Secreted Acetylcholinesterase Revealed by Heterologous Expression in Trypanosoma musculi.

Nematode parasites secrete molecules which regulate the mammalian immune system, but their genetic intractability is a major impediment to identifying and characterising the biological effects of these molecules. We describe here a novel system for heterologous expression of helminth secreted proteins in the natural parasite of mice, Trypanosoma musculi, which can be used to analyse putative immunomodulatory functions. Trypanosomes were engineered to express a secreted acetylcholinesterase from Nippostrongylus brasiliensis. Infection of mice with transgenic parasites expressing acetylcholinesterase resulted in truncated infection, with trypanosomes cleared early from the circulation. Analysis of cellular phenotypes indicated that exposure to acetylcholinesterase in vivo promoted classical activation of macrophages (M1), with elevated production of nitric oxide and lowered arginase activity. This most likely occurred due to the altered cytokine environment, as splenocytes from mice infected with T. musculi expressing acetylcholinesterase showed enhanced production of IFNγ and TNFα, with diminished IL-4, IL-13 and IL-5. These results suggest that one of the functions of nematode secreted acetylcholinesterase may be to alter the cytokine environment in order to inhibit development of M2 macrophages which are deleterious to parasite survival. Transgenic T. musculi represents a valuable new vehicle to screen for novel immunoregulatory proteins by extracellular delivery in vivo to the murine host.

Comparative analysis of the interaction of Helicobacter pylori with human dendritic cells, macrophages, and monocytes.

Helicobacter pylori may cause chronic gastritis, gastric cancer, or lymphoma. Myeloid antigen-presenting cells (APCs) are most likely involved in the induction and expression of the underlying inflammatory responses. To study the interaction of human APC subsets with H. pylori, we infected monocytes, monocyte-derived dendritic cells (DCs), and monocyte-derived (classically activated; M1) macrophages with H. pylori and analyzed phenotypic alterations, cytokine secretion, phagocytosis, and immunostimulation. Since we detected CD163(+) (alternatively activated; M2) macrophages in gastric biopsy specimens from H. pylori-positive patients, we also included monocyte-derived M2 macrophages in the study. Upon H. pylori infection, monocytes secreted interleukin-1ß (IL-1ß), IL-6, IL-10, and IL-12p40 (partially secreted as IL-23) but not IL-12p70. Infected DCs became activated, as shown by the enhanced expression of CD25, CD80, CD83, PDL-1, and CCR7, and secreted IL-1ß, IL-6, IL-10, IL-12p40, IL-12p70, and IL-23. However, infection led to significantly downregulated CD209 and suppressed the constitutive secretion of macrophage migration inhibitory factor (MIF). H. pylori-infected M1 macrophages upregulated CD14 and CD32, downregulated CD11b and HLA-DR, and secreted mainly IL-1ß, IL-6, IL-10, IL-12p40, and IL-23. Activation of DCs and M1 macrophages correlated with increased capacity to induce T-cell proliferation and decreased phagocytosis of dextran. M2 macrophages upregulated CD14 and CD206 and secreted IL-10 but produced less of the proinflammatory cytokines than M1 macrophages. Thus, H. pylori affects the functions of human APC subsets differently, which may influence the course and the outcome of H. pylori infection. The suppression of MIF in DCs constitutes a novel immune evasion mechanism exploited by H. pylori.

Unity and diversity of metacognition.

Despite many research efforts dedicated toward deciphering the functional architecture underlying metacognition, it is still unclear if there is a common metacognitive resource for different functional requirements. Here, using laboratory measures of metacognition across several domains in a large sample (N = 155), we examined whether metacognitive ability is determined by universal or modular processes, and whether "online" laboratory measures are related to "offline" self-report measures of real-world metacognition. Trial-by-trial ratings of confidence were collected in pairs of tasks tapping into the domains of visual perception and episodic memory, whereas in the attention-to-action domain, one task obtained trial-by-trial confidence ratings and the other signal-dependent measures of error awareness. Relationships between metacognitive efficiency scores across paradigms and domains were assessed using a combination of correlational and latent variable approaches. The results point to a mixture of domain-general (unity) and domain-specific (diversity) components. Specifically, Bayesian correlation estimates of metacognitive efficiency as well as confirmatory factor analysis of interdomain correlations suggested metacognition about perceptual judgments to be mostly domain-specific, whereas convergent indications for interrelations between metacognition in the domains of attention-to-action and memory implied the coexistence of partly specialized metacognitive subsystems. Notably, offline measures of metacognition represented online metacognitive bias rather than online metacognitive efficiency, underscoring prevalent skepticism whether self-report questionnaires provide a useful proxy in metacognition research, as they appear susceptible to potentially unreliable introspections and memory distortions. Overall, our results indicate a constitution of both universal and specialized parts for task-based metacognition. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Structural alterations of the insula in depression patients - A 7-Tesla-MRI study.

INTRODUCTION: The insular cortex is part of a network of highly connected cerebral "rich club" - regions and has been implicated in the pathophysiology of various psychiatric and neurological disorders, of which major depressive disease is one of the most prevalent. "Rich club" vulnerability can be a contributing factor in disease development. High-resolution structural subfield analysis of insular volume in combination with cortical thickness measurements and psychological testing might elucidate the way in which the insula is changed in depression. MATERIAL AND METHODS: High-resolution structural images of the brain were acquired using a 7T-MRI scanner. The mean grey matter volume and cortical thickness within the insular subfields were analysed using voxel-based morphometry (VBM) and surface analysis techniques respectively. Insular subfields were defined according to the Brainnetome Atlas for VBM - and the Destrieux-Atlas for cortical thickness - analysis. Thirty-three patients with confirmed major depressive disease, as well as thirty-one healthy controls matched for age and gender, were measured. The severity of depression in MDD patients was measured via a BDI-II score and objective clinical assessment (AMDP). Intergroup statistical analysis was performed using ANCOVA. An intragroup multivariate regression analysis of patient psychological test results was calculated. Corrections for multiple comparisons was performed using FDR. RESULTS: Significant differences between groups were observed in the left granular dorsal insula according to VBM-analysis. AMDP-scores positively correlated with cortical thickness in the right superior segment of the circular insular sulcus. CONCLUSIONS: The combination of differences in grey matter volume between healthy controls and patients with a positive correlation of cortical thickness with disease severity underscores the insula's role in the pathogeneses of MDD. The connectivity hub insular cortex seems vulnerable to disruption in context of affective disease.

Trichinella spiralis secretes abundant unencapsulated small RNAs with potential effects on host gene expression.

Many organisms, including parasitic nematodes, secrete small RNAs into the extracellular environment, largely encapsulated within small vesicles. Parasite-secreted material often contains microRNAs (miRNAs), raising the possibility that they might regulate host genes in target cells. Here we characterise secreted RNAs from the parasitic nematode Trichinella spiralis at two different life stages. We show that adult T. spiralis, which inhabit intestinal mucosa, secrete miRNAs within vesicles. Unexpectedly, T. spiralis muscle stage larvae, which live intracellularly within skeletal muscle cells, secrete miRNAs that appear not to be encapsulated. Notably, secreted miRNAs include a homologue of mammalian miRNA-31, which has an important role in muscle development. Our work therefore suggests that RNAs may be secreted without encapsulation in vesicles, with implications for the biology of T. spiralis infection.

Taenia larvae possess distinct acetylcholinesterase profiles with implications for host cholinergic signalling.

Larvae of the cestodes Taenia solium and Taenia crassiceps infect the central nervous system of humans. Taenia solium larvae in the brain cause neurocysticercosis, the leading cause of adult-acquired epilepsy worldwide. Relatively little is understood about how cestode-derived products modulate host neural and immune signalling. Acetylcholinesterases, a class of enzyme that breaks down acetylcholine, are produced by a host of parasitic worms to aid their survival in the host. Acetylcholine is an important signalling molecule in both the human nervous and immune systems, with powerful modulatory effects on the excitability of cortical networks. Therefore, it is important to establish whether cestode derived acetylcholinesterases may alter host neuronal cholinergic signalling. Here we make use of multiple techniques to profile acetylcholinesterase activity in different extracts of both Taenia crassiceps and Taenia solium larvae. We find that the larvae of both species contain substantial acetylcholinesterase activity. However, acetylcholinesterase activity is lower in Taenia solium as compared to Taenia crassiceps larvae. Further, whilst we observed acetylcholinesterase activity in all fractions of Taenia crassiceps larvae, including on the membrane surface and in the excreted/secreted extracts, we could not identify acetylcholinesterases on the membrane surface or in the excreted/secreted extracts of Taenia solium larvae. Bioinformatic analysis revealed conservation of the functional protein domains in the Taenia solium acetylcholinesterases, when compared to the homologous human sequence. Finally, using whole-cell patch clamp recordings in rat hippocampal brain slice cultures, we demonstrate that Taenia larval derived acetylcholinesterases can break down acetylcholine at a concentration which induces changes in neuronal signalling. Together, these findings highlight the possibility that Taenia larval acetylcholinesterases can interfere with cholinergic signalling in the host, potentially contributing to pathogenesis in neurocysticercosis.

Knocking down schistosomes - promise for lentiviral transduction in parasites.

Underpinned by major advances in our understanding of the genomes of schistosomes, progress in the development of functional genomic tools is providing unique prospects to gain insights into the intricacies of the biology of these blood flukes, their host relationships, and the diseases that they cause. This article reviews some key applications of double-stranded RNA interference (RNAi) in Schistosoma mansoni, appraises delivery systems for transgenesis and stable gene silencing, considers ways of increasing efficiency and specificity of gene silencing, and discusses the prospects of using a lentivirus delivery system for future functional genomic-phenomic explorations of schistosomes and other parasites. The ability to achieve effective and stable gene perturbation in parasites has major biological implications and could facilitate the development of new interventions.

Prospects for Vector-Based Gene Silencing to Explore Immunobiological Features of Schistosoma mansoni.

Schistosomiasis is a prevalent, socioeconomically important disease of humans caused by parasites of the genus Schistosoma (schistosomes or blood flukes). Currently, more than 200 million people worldwide are infected with schistosomes. Despite major research efforts, there is only one drug routinely used for effective treatment, and no vaccine is available to combat schistosomiasis. The purpose of the present article is to (1) provide a background on the parasites and different forms of disease; (2) describe key immunomolecular aspects of disease induced in the host; and (3) critically appraise functional genomic methods employed to explore parasite biology, parasite-host interactions and disease at the molecular level. Importantly, the article also describes the features and advantages of lentiviral delivery of artificial microRNAs to silence genes. It also discusses the first successful application of such an approach in schistosomes, in order to explore the immunobiological role of selected target proteins known to be involved in egg-induced disease. The lentiviral transduction system provides exciting prospects for future, fundamental investigations of schistosomes, and is likely to have broad applicability to other eukaryotic pathogens and infectious diseases. The ability to achieve effective and stable gene perturbation in parasites has major biotechnological implications, and might facilitate the development of radically new methods for the treatment and control of parasitic diseases.

Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo.

Schistosomiasis, one of the most important neglected tropical diseases worldwide, is caused by flatworms (blood flukes or schistosomes) that live in the bloodstream of humans. The hepatointestinal form of this debilitating disease results from a chronic infection with Schistosoma mansoni or Schistosoma japonicum. No vaccine is available to prevent schistosomiasis, and treatment relies predominantly on the use of a single drug, praziquantel. In spite of considerable research effort over the years, very little is known about the complex in vivo events that lead to granuloma formation and other pathological changes during infection. Here we use, for the first time, a lentivirus-based transduction system to deliver microRNA-adapted short hairpin RNAs (shRNAmirs) into the parasite to silence and explore selected protein-encoding genes of S. mansoni implicated in the disease process. This gene-silencing system has potential to be used for functional genomic-phenomic studies of a range of socioeconomically important pathogens.

The effect of music on 10-km cycle time-trial performance.

Music is widely used as an ergogenic aid in sport, but there is little evidence of its effectiveness during closed-loop athletic events. In order to determine the effectiveness of music as an ergogenic aid, well-trained and task-habituated cyclists performed 10-km cycle time trials either while listening to self-selected motivational music or with auditory input blocked. There were no statistically significant differences in performance time or physiological or psychological markers related to music (time-trial duration17.75 ± 2.10 vs 17.81 ± 2.06 min, mean power output 222 ± 66 vs 220 ± 65 W, peak heart rate184 ± 9 vs 183 ± 8 beats/min, peak blood lactate12.1 ± 2.6 vs 11.9 ± 2.1 mmol/L, and final rating of perceived exertion 8.4 ± 1.5 vs 8.5 ± 1.6). It is concluded that during exercise at competitive intensity, there is no meaningful effect of music on either performance or physiology.

Calreticulin from the intestinal nematode Heligmosomoides polygyrus is a Th2-skewing protein and interacts with murine scavenger receptor-A.

Helminth infections are commonly associated with a Th2 immune response, yet only a few parasite molecules involved in triggering such immune responses have been identified. Here, we describe the Th2-skewing property of calreticulin of Heligmosomoides polygyrus (HpCRT). HpCRT is a secreted protein most abundantly expressed by tissue invasive larvae (L4). Native HpCRT purified from adult worm extract (nHpCRT) stimulated robust IL-4 release from CD4(+) T cells of H. polygyrus infected mice. Interestingly, CD4(+) T cells also produced significant amounts of IL-10 while IFN-gamma was not detectable. Likewise, immunization with recombinant HpCRT (rHpCRT) without extrinsic adjuvant led predominantly to a specific IL-4 production implying the innate ability of HpCRT to drive Th2 responses. The triggering of a Th2-skewed immune response to rHpCRT is corroborated by the induction of HpCRT-specific IgG1 and IgE antibodies. Furthermore, rHpCRT bound to scavenger receptor type A (SR-A) on dendritic cells, and interaction of HpCRT with SR-A led to internalization of HpCRT that could be partially blocked by competition with SR-A ligands as well as with an anti-SR-A monoclonal antibody. Hence, our data imply that nematode calreticulin interacts with a mammalian scavenger receptor and at the same time induces a Th2 response.

Double gametocyte infections in apicomplexan parasites of birds and reptiles.

The simultaneous occurrence of male and female gametocytes inside a single host blood cell has been suggested to enhance apicomplexan transmission ["double gametocyte infection (DGI) hypothesis"]. We did a bibliographic search and a direct screen of blood smears from wild birds and reptiles to answer, for the first time, how common are these infections in the wild. Taking these two approaches together, we report here cases of DGIs in Plasmodium, Haemoproteus, Leucocytozoon and Hepatozoon, and cases of male-female DGIs in Haemoproteus of birds and reptiles and in Leucocytozoon of birds. Thus, we suggest that DGIs and male female DGIs are more widespread than previously thought, opening a new research avenue on apicomplexan transmission.