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HDV infection causes severe liver disease, the global health burden of which may be underestimated due to limited epidemiological data. HDV depends on HBV for infection, but recent studies indicated that dissemination can also be supported by other helper viruses such as HCV. We used a rapid point-of-care test and an ELISA to retrospectively test for antibodies against the Hepatitis Delta antigen (anti-HDV-Ab) in 4103 HBsAg-positive and 1661 HBsAg-negative, anti-HCV-positive sera from China and Germany. We found that the HDV seroprevalence in HBsAg-positive patients in China is limited to geographic hotspots (Inner Mongolia: 35/251, 13.9%, Xinjiang: 7/180, 3.9%) and high-risk intravenous drug users (HBV mono-infected: 23/247, 9.3%, HBV-HCV co-infected: 34/107, 31.8%), while none of the 2634 HBsAg carriers from other metropolitan regions were anti-HDV-Ab-positive. In Germany, we recorded an HDV seroprevalence of 5.3% in a university hospital environment. In a cohort of HBsAg-negative, anti-HCV-positive patients that were not exposed to HBV before (anti-HBc-negative), HDV was not associated with HCV mono-infection (Chinese high-risk cohort: 0/365, 0.0%, German mixed cohort: 0/263, 0.0%). However, 21/1033 (2.0%) high-risk HCV patients in China with markers of a previously cleared HBV infection (anti-HBc-positive) were positive for anti-HDV-Ab, with two of them being positive for both HDV and HCV RNA but negative for HBV DNA. The absence of anti-HDV-Ab in HCV mono-infected patients shows that HCV cannot promote HDV transmission in humans.

Background & Aims Chronic HDV infections cause the most severe form of viral hepatitis. HDV requires HBV envelope proteins for hepatocyte entry, particle assembly and release. Eight HDV and 8 HBV genotypes have been identified. However, there are limited data on the replication competence of different genotypes and the effect that different HBV envelopes have on virion assembly and infectivity. Methods We subcloned complementary DNAs (cDNAs) of all HDV and HBV genotypes and systematically studied HDV replication, assembly and infectivity using northern blot, western blot, reverse-transcription quantitative PCR, and in-cell ELISA. Results The 8 HDV cDNA clones initiated HDV replication with noticeable differences regarding replication efficacy. The 8 HBV-HBsAg-encoding constructs all supported secretion of subviral particles, however variations in envelope protein stoichiometry and secretion efficacy were observed. Co-transfection of all HDV/HBV combinations supported particle assembly, however, the respective pseudo-typed HDVs differed with respect to assembly kinetics. The most productive combinations did not correlate with the natural geographic distribution, arguing against an evolutionary adaptation of HDV ribonucleoprotein complexes to HBV envelopes. All HDVs elicited robust and comparable innate immune responses. HBV envelope-dependent differences in the activity of the EMA-approved entry inhibitor bulevirtide were observed, however efficient inhibition could be achieved at therapeutically applied doses. Lonafarnib also showed pan-genotypic activity. Conclusions HDVs from different genotypes replicate with variable efficacies. Variations in HDV genomes and HBV envelope proteins are both major determinants of HDV egress and entry efficacy, and consequently assembly inhibition by lonafarnib or entry inhibition by bulevirtide. These differences possibly influence HDV pathogenicity, immune responses and the efficacy of novel drug regimens. Lay summary HDV requires the envelope protein of HBV for assembly and to infect human cells. We investigated the ability of different HDV genotypes to infect cells and replicate. We also assessed the effect that envelope proteins from different HBV genotypes had on HDV infectivity and replication. Herein, we confirmed that genotypic differences in HDV and HBV envelope proteins are major determinants of HDV assembly, de novo cell entry and consequently the efficacy of novel antivirals.

Controlling invasive species is critical for conservation but can have unintended consequences for native species and divert resources away from other efforts. This dilemma occurs on a grand scale in the North American Great Lakes, where dams and culverts block tributary access to habitat of desirable fish species and are a lynchpin of long-standing efforts to limit ecological damage inflicted by the invasive, parasitic sea lamprey (Petromyzon marinus). Habitat restoration and sea-lamprey control create conflicting goals for managing aging infrastructure. We used optimization to minimize opportunity costs of habitat gains for 37 desirable migratory fishes that arose from restricting sea lamprey access (0-25% increase) when selecting barriers for removal under a limited budget (US$1-105 million). Imposing limits on sea lamprey habitat reduced gains in tributary access for desirable species by 15-50% relative to an unconstrained scenario. Additional investment to offset the effect of limiting sea-lamprey access resulted in high opportunity costs for 30 of 37 species (e.g., an additional US$20-80 million for lake sturgeon [Acipenser fulvescens]) and often required ≥5% increase in sea-lamprey access to identify barrier-removal solutions adhering to the budget and limiting access. Narrowly distributed species exhibited the highest opportunity costs but benefited more at less cost when small increases in sea-lamprey access were allowed. Our results illustrate the value of optimization in limiting opportunity costs when balancing invasion control against restoration benefits for diverse desirable species. Such trade-off analyses are essential to the restoration of connectivity within fragmented rivers without unleashing invaders.

Rapid evolution of pest, pathogen, and wildlife populations can have undesirable effects, for example, when insects evolve resistance to pesticides or fishes evolve smaller body size in response to harvest. A destructive invasive species in the Laurentian Great Lakes, the sea lamprey (Petromyzon marinus) has been controlled with the pesticide 3-trifluoromethyl-4-nitrophenol (TFM) since the 1950s. We evaluated the likelihood of sea lamprey evolving resistance to TFM by (i) reviewing sea lamprey life history and control; (ii) identifying physiological and behavioural resistance strategies; (iii) estimating the strength of selection from TFM; (iv) assessing the timeline for evolution; and (v) analyzing historical toxicity data for evidence of resistance. The number of sea lamprey generations exposed to TFM was within the range observed for fish populations where rapid evolution has occurred. Mortality from TFM was estimated as 82%–90%, suggesting significant selective pressure. However, 57 years of toxicity data revealed no increase in lethal concentrations of TFM. Vigilance and the development of alternative controls are required to prevent this aquatic invasive species from evolving strategies to evade control.

Background Equine gastrointestinal nematodes (GINs) have been the subject of intermittent studies in Australia over the past few decades. However, comprehensive information on the epidemiology of equine GINs, the efficacy of available anthelmintic drugs and the prevalence of anthelmintic resistance (AR) in Australasia is lacking. Herein, we have systematically reviewed existing knowledge on the horse GINs recorded in Australia, and main aspects of their pathogeneses, epidemiology, diagnoses, treatment and control. Methods Six electronic databases were searched for publications on GINs of Australian horses that met our inclusion criteria for the systematic review. Subsets of publications were subjected to review epidemiology, diagnoses, pathogeneses, treatment and control of GINs of horses from Australia. Results A total of 51 articles published between 1950 to 2018 were included. The main GINs reported in Australian horses were cyathostomins (at least 28 species), Draschia megastoma, Habronema muscae, H. majus, Oxyuris equi, Parascaris equorum, Strongyloides westeri and Trichostrongylus axei across different climatic regions of Queensland, New South Wales, Victoria, and Western Australia. Nematodes are diagnosed based on the traditional McMaster egg counting technique, though molecular markers to characterise common GINs of equines were characterised in 1990s. The use of anthelmintic drugs remains the most widely-used strategy for controlling equine GIN parasites in Australia; however, the threshold of faecal egg count that should trigger treatment in horses, remains controversial. Furthermore, anthelmintic resistance within GIN population of horses is becoming a common problem in Australia. Conclusions Although GINs infecting Australian horses have been the subject of occasional studies over the past few decades, the effective control of GIN infections is hampered by a generalised lack of knowledge in various disciplines of equine parasitology. Therefore, coordinated and focused research is required to fill our knowledge gaps in these areas to maximise equine health and minimise economic losses associated with the parasitic infections in Australia. Electronic supplementary material The online version of this article (10.1186/s13071-019-3445-4) contains supplementary material, which is available to authorized users.

Microglial cells, the resident macrophage population of the central nervous system (CNS), actively scan tissue under both normal and pathologic contexts. Their resulting engagement can become either neuroprotective or neurotoxic, leading to amelioration or aggravation of disease progression. In this review, we focus on the molecular signaling molecules involved in microglial responses and discuss observations demonstrating the diverse effects of microglia in animal models of CNS diseases.

Focal cerebral ischemia and traumatic brain injury induce an escalating amount of cell death because of harmful mediators diffusing from the original lesion site. Evidence suggests that healthy cells surrounding these lesions attempt to protect themselves by producing endocannabinoids (eCBs) and activating cannabinoid receptors, the molecular target for marijuana-derived compounds. Indeed, activation of cannabinoid receptors reduces the production and diffusion of harmful mediators. Here, we provide evidence that an exception to this pattern is found in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We show that cell damage induced by EAE does not lead to increase in eCBs, even though cannabinoid receptors are functional because synthetic cannabinoid agonists are known to confine EAE-induced lesions. This lack of eCB increase is likely due to IFN-γ, which is released by primed T cells invading the CNS. We show that IFN-γ disrupts the functionality of purinergic P2X 7 receptors, a key step controlling eCB production by microglia, the main source of eCBs in brain. Accordingly, induction of EAE in P2X 7 −/− mice results in even lower eCB levels and more pronounced cell damage than in wild-type mice. Our data suggest that the high level of CNS IFN-γ associated with EAE disrupts eCB-mediated neuroprotection while maintaining functional cannabinoid receptors, thus providing additional support for the use of cannabinoid-based medicine to treat multiple sclerosis.

The cytoplasm of neural cells contain millimolar amounts of ATP, which flood the extracellular space after injury, activating purinergic receptors expressed by glial cells and increasing gliotransmitter production. These gliotransmitters, which are thought to orchestrate neuroinflammation, remain widely uncharacterized. Recently, we showed that microglial cells produce 2-arachidonoylglycerol (2-AG), an endocannabinoid known to prevent the propagation of harmful neuroinflammation, and that ATP increases this production by threefold at 2.5 min (Witting et al., 2004). Here we show that ATP increases 2-AG production from mouse astrocytes in culture, a response that is more rapid (i.e., significant within 10 sec) and pronounced (i.e., 60-fold increase at 2.5 min) than any stimulus-induced increase in endocannabinoid production reported thus far. Increased 2-AG production from astrocytes requires millimolar amounts of ATP, activation of purinergic P2X7receptors, sustained rise in intracellular calcium, and diacylglycerol lipase activity. Furthermore, we show that astrocytes express monoacylglycerol lipase (MGL), the main hydrolyzing enzyme of 2-AG, the pharmacological inhibition of which potentiates the ATP-induced 2-AG production (up to 113-fold of basal 2-AG production at 2.5 min). Our results show that ATP greatly increases, and MGL limits, 2-AG production from astrocytes. We propose that 2-AG may function as a gliotransmitter, with MGL inhibitors potentiating this production and possibly restraining the propagation of harmful neuroinflammation.

Growing evidence suggests that a major physiological function of the cannabinoid signaling system is to modulate neuroinflammation. This review discusses the anti-inflammatory properties of cannabinoid compounds at molecular, cellular and whole animal levels, first by examining the evidence for anti-inflammatory effects of cannabinoids obtained using in vivo animal models of clinical neuroinflammatory conditions, specifically rodent models of multiple sclerosis, and second by describing the endogenous cannabinoid (endocannabinoid) system components in immune cells. Our aim is to identify immune functions modulated by cannabinoids that could account for their anti-inflammatory effects in these animal models.

Astrocytes play an important role in neuroprotective responses. Recent studies indicate that endothelin-1, a neuropeptide upregulated during brain injury, increases levels of the endocannabinoid anandamide, a lipid with neuroprotective properties, in astrocytes in primary cultures. However, whether this neuropeptide also alters levels of 2-arachidonoyl glycerol (2-AG), the most abundant endocannabinoid in the CNS, in astrocytes remains unknown. In addition, 2-AG levels in astrocytes have never been measured. In this report we use chemical ionization gas chromatography/mass spectrometry to quantify picomole amounts of 2-AG in primary cultures of mouse astrocytes. We also demonstrate that endothelin-1 increases 2-AG production by 5-fold in these cells, a response that requires extracellular calcium and endothelin-1(A) receptor engagement. Immunocytochemistry showed that although cultured mouse neurons and microglia express cannabinoid receptors, cultured astrocytes do not. The data suggest that endothelin-1 modulates 2-AG production in astrocytes and that this endocannabinoid may participate in paracrine signaling toward neurons and microglia.

During neuroinflammation, activated microglial cells migrate toward dying neurons, where they exacerbate local cell damage. The signaling molecules that trigger microglial cell migration are poorly understood. In this paper, we show that pathological overstimulation of neurons by glutamate plus carbachol dramatically increases the production of the endocannabinoid 2-arachidonylglycerol (2-AG) but only slightly increases the production of anandamide and does not affect the production of two putative endocannabinoids, homo-gamma-linolenylethanolamide and docosatetraenylethanolamide. We further show that pathological stimulation of microglial cells with ATP also increases the production of 2-AG without affecting the amount of other endocannabinoids. Using a Boyden chamber assay, we provide evidence that 2-AG triggers microglial cell migration. This effect of 2-AG occurs through CB2 and abnormal-cannabidiol-sensitive receptors, with subsequent activation of the extracellular signal-regulated kinase 1/2 signal transduction pathway. It is important to note that cannabinol and cannabidiol, two nonpsychotropic ingredients present in the marijuana plant, prevent the 2-AG-induced cell migration by antagonizing the CB2 and abnormal-cannabidiol-sensitive receptors, respectively. Finally, we show that microglial cells express CB2 receptors at the leading edge of lamellipodia, which is consistent with the involvement of microglial cells in cell migration. Our study identifies a cannabinoid signaling system regulating microglial cell migration. Because this signaling system is likely to be involved in recruiting microglial cells toward dying neurons, we propose that cannabinol and cannabidiol are promising nonpsychotropic therapeutics to prevent the recruitment of these cells at neuroinflammatory lesion sites.

Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 109 particles/ml for 1 h incorporated an average of 3.39 × 108 Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 105 counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific 154Sm-tagged anti–HLA-DR or 174Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

The CNS is considered immune privileged due to the blood-brain barrier and the absence of conventional lymphatics. Nonetheless, T cell immune responses specific for CNS Ag have been documented. Where these events are initiated and what cellular mechanisms are involved remain unknown. In this study, we established an experimental mouse model to evaluate the requirements for priming CD8+ T cells following the cross-presentation of intracranial Ag. Surprisingly, we find that even with a damaged blood-brain barrier, Ag presentation occurs in regional lymph nodes and not within the CNS itself. Only once the responding cells have expanded can they traffic to the site of CNS injury. Cross-presentation of intracranial Ag is efficient and the subsequent priming of CD8+ T cells is dependent on CD4+ T cell help and CD40 signaling in host APCs. Our findings have important implications for the initiation of T cell immune responses toward CNS Ags.

Endogenous cannabinoid ligands (endocannabinoids) produced by neurons, astrocytes, and microglial cells activate cannabinoid receptors, the molecular target for marijuana's bioactive ingredient Δ 9 -tetrahydrocannabinol. The molecular mechanism underlying the production of the most abundant endocannabinoid, 2-arachidonoylglycerol (2-AG), is unclear. A prevalent hypothesis proposes that activation of metabotropic receptors coupled to the phosphatidylinositol-specific phospholipase C and diacylglycerol (DG) lipase pathway will systematically lead to increases in 2-AG production. Here, we show that ATP increases 2-AG production by cultured microglial cells in a phosphatidylinositol-specific phospholipase C and DG lipase-dependent manner. However, efficacious activation of metabotropic P2Y purinergic receptors coupled to phosphatidylinositol-specific phospholipase C does not increase 2-AG production. This suggests that ionotropic, and not metabotropic, purinergic receptors control 2-AG production at an unexpected enzymatic step of its metabolic pathway. We show that activation of P2X 7 ionotropic receptors, which are highly permeable to calcium, is necessary and sufficient to increase 2-AG production in microglial cells. We also show that the sustained rise in intracellular calcium induced by activation of P2X 7 receptors directly increases DG lipase activity while inhibiting the activity of monoacylglycerol lipase, the enzyme that degrades 2-AG. This inverse sensitivity of DG lipase and monoacylglycerol lipase to calcium constitutes an original and efficient modality for sustained accumulation of 2-AG. Because prolonged increases in 2-AG amounts in brain parenchyma are thought to orchestrate neuroinflammation, the enzymatic steps involved in 2-AG synthesis and degradation by microglial cells constitute appealing targets for therapy aimed at controlling exacerbated neuroinflammation.

Focal cerebral ischemia (FCI) induces rapid neuronal death in the ischemic core, which gradually expands toward the penumbra, partly as the result of a neuroinflammatory response. It is known that propagation of neuroinflammation involves microglial cells, the resident macrophages of the brain, which are highly motile when activated by specific signals. However, the signals that increase microglial cell motility in response to FCI remain mostly elusive.Here, we tested the hypothesis that endocannabinoids mediate neuroinflammation propagation by increasing microglial cell motility. We found that, in mouse cerebral cortex, FCI greatly increases palmitoylethanolamide (PEA), only moderately increases anandamide [arachidonylethanolamide (AEA)], and does not affect 2-arachidonoylglycerol levels. We also found that PEA potentiates AEA-induced microglial cell migration, without affecting other steps of microglial activation, such as proliferation, particle engulfment, and nitric oxide production. This potentiation of microglial cell migration by PEA involves reduction in cAMP levels. In line with this, we provide evidence that PEA acts through Gi/o-coupled receptors. Interestingly, these receptors engaged by PEA are pharmacologically distinct from CB1and CB2cannabinoid receptors, as well as from the WIN and abn-CBD (abnormal-cannabidiol) receptors, two recently identified cannabinoid receptors.Our results show that PEA and AEA increase after FCI and synergistically enhance microglial cell motility. Because such a response could participate in the propagation of the FCI-induced neuroinflammation within the CNS, and because PEA is likely to act through its own receptor, a better understanding of the receptor engaged by PEA may help guide the search for improved therapies against neuroinflammation.

Anandamide (arachidonylethanolamide) is an endocannabinoid that belongs to the acylethanolamide lipid family. It is produced by neurons in a calcium-dependent manner and acts through cannabinoid CB1 receptors. Other members of the acylethanolamide lipid family are also produced by neurons and act through G-protein-coupled receptors: homo-gamma-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA) act through CB1 receptors, palmitylethanolamide (PEA) acts through CB2-like receptors, and oleylethanolamide (OEA) acts through receptors that have not yet been cloned. Although it is clear that anandamide and other acylethanolamides play a major role in neuronal signaling, whether astrocytes also produce these lipids is unknown. We developed a chemical ionization gas chromatography/mass spectrometry method that allows femtomole detection and quantification of anandamide and other acylethanolamides. Using this method, we unambiguously detected and quantified anandamide, HEA, DEA, PEA, and OEA in mouse astrocytes in culture. Stimulation of mouse astrocytes with ionomycin, a calcium ionophore, enhanced the production of anandamide, HEA, and DEA, whereas PEA and OEA levels were unchanged. Endothelin-1, a peptide known to act on astrocytes, enhanced the production of anandamide, without affecting the levels of other acylethanolamides. These results show that astrocytes produce anandamide, HEA, and DEA in a calcium-dependent manner and that anandamide biosynthesis can be selectively stimulated under physiologically relevant conditions. The relative levels of acylethanolamides in astrocytes from rat and human were different from the relative levels of acylethanolamides in mouse astrocytes, indicating that the production of these lipids differs between species. Because astrocytes are known to express CB1 receptors and inactivate endocannabinoids, our finding strongly suggests the existence of a functional endocannabinoid signaling system in these cells.