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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.

Background/aim ALK inhibitors like Crizotinib, Ceritinib and Alectinib are targeted therapies used in patients with anaplastic lymphoma kinase (ALK)-positive, advanced non-small cell lung cancer (NSCLC). Since in this tumor entity radiotherapy is employed sequentially or concomitantly, potential synergistic effects were investigated, which may support the hypothesis of induced radiosensitization by using ALK inhibitors. Materials and methods Two cell lines expressing wild-type (WT) or echinoderm microtubule-associated protein-like 4 (EML4)-ALK were treated with ALK inhibitors, followed by irradiation. Cell survival, cell death, cell cycle and phosphorylation of H2A histone family, member X (H2AX) were examined. Results Combined treatment with ALK-inhibitors plus 10 Gy-irradiation led to effects similar to those of sole radiotherapy, but was more effective than sole drug treatment. Conclusion There is no clear evidence of sensitization to radiation by treating EML4-ALK mutated cells with ALK inhibitors.

Cellular phenotypes are established and controlled by complex and precisely orchestrated molecular networks. In cancer, mutations and dysregulations of multiple molecular factors perturb the regulation of these networks and lead to malignant transformation. High-throughput technologies are a valuable source of information to establish the complex molecular relationships behind the emergence of malignancy, but full exploitation of this massive amount of data requires bioinformatics tools that rely on network-based analyses. In this report we present the Virtual Melanoma Cell, an online tool developed to facilitate the mining and interpretation of high-throughput data on melanoma by biomedical researches. The platform is based on a comprehensive, manually generated and expert-validated regulatory map composed of signaling pathways important in malignant melanoma. The Virtual Melanoma Cell is a tool designed to accept, visualize and analyze user-generated datasets. It is available at: https://www.vcells.net/melanoma . To illustrate the utilization of the web platform and the regulatory map, we have analyzed a large publicly available dataset accounting for anti-PD1 immunotherapy treatment of malignant melanoma patients.

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.

Background/aim Recent evidence suggests that melanoma patients treated with BRAF inhibitors experience radiosensitization with an increased frequency of side-effects. This could also imply increased effectiveness when treating melanoma. Materials and methods To test whether the BRAF inhibitors dabrafenib and vemurafenib together with ionizing radiation more effectively inhibit melanoma cells, primary human melanoma tumor cell lines expressing wild-type (WT) or mutant V600E BRAF were analyzed by cell survival, cell death, and cell-cycle testing. Results All melanoma cell lines examined were radioresistant in these assays. BRAF inhibitor treatment alone suppressed cell survival more effectively than radiation in all the mutant V600E BRAF cell lines, and vemurafenib, but not dabrafenib, also inhibited cell survival in the WT BRAF cell lines at clinically relevant concentrations. However, when cells were treated with BRAF inhibitor followed by radiation, there was no increased effect on the suppression of cell survival. Vemurafenib induced more necrosis than radiation in most melanoma cell lines, irrespective of BRAF status, but this effect was not additive with the combination treatment. BRAF inhibitors and radiation had variable, but independent effects on the induction of cell-cycle arrest. Conclusion These results suggest that BRAF inhibitors and ionizing radiation do not act synergistically to inhibit the growth of primary human melanoma cells.

Administering immunoregulatory cells as medicinal agents is a revolutionary approach to the treatment of immunologically mediated diseases. Isolating, propagating, and modifying cells before applying them to patients allows complementation of specific cellular functions, which opens astonishing new possibilities for gain-of-function antigen-specific treatments in autoimmunity, chronic inflammatory disorders, and transplantation. This critical review presents a systematic assessment of the potential clinical risks posed by cell-based immunotherapy, focusing on treatment of renal transplant recipients with regulatory macrophages as a concrete example.

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.

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.

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.