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Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs. M. G.-L. was a postdoctoral fellow supported by the Plan Estratégico de Investigación e Innovación en Salud (PERIS, SLT002/16/00179) of the Generalitat de Catalunya, Spain. M. D.-V. was a predoctoral fellow supported by Secretaria d’Universitats i Recerca del Departament d’Economia i Creixement, Generalitat de Catalunya (2017 FI_B2_00029). I. A.-H. is a predoctoral fellow supported by the Ministerio de Economia y Competitividad (FPI BES-2017081657). C. S. was funded by the German Research Foundation (DFG; fellowship SCHA2047/1-1). We acknowledge support from the National Institute of Health R21 program (1R21AI135680-01). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech), and it is supported by “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2017SGR595) and acknowledges support of the Spanish Ministry of Science and Innovation to the EMBL partnership. We also acknowledge support from the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S) and support from the Generalitat de Catalunya through the CERCA Program. This research is part of the ISGlobal’s Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces. Work in the laboratory of Carmen Fernandez-Becerra and Hernando A. del Portillo is funded by the Ministerio Español de Economía y Competitividad (SAF2016 80655-R) and by and by the Ministerio de Ciencia e Innovación (PID2019-111795RB-I00). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Following their inoculation by the bite of an infected Anopheles mosquito, the malaria parasite sporozoite forms travel from the bite site in the skin into the bloodstream, which transports them to the liver. The thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that is released from secretory organelles and relocalized on the sporozoite plasma membrane. TRAP is required for sporozoite motility and host infection, and its extracellular portion contains adhesive domains that are predicted to engage host receptors. Here, we identified the human platelet-derived growth factor receptor β (hPDGFRβ) as one such protein receptor. Deletion constructs showed that the von Willebrand factor type A and thrombospondin repeat domains of TRAP are both required for optimal binding to hPDGFRβ-expressing cells. We also demonstrate that this interaction is conserved in the human-infective parasite Plasmodium vivax, but not the rodent-infective parasite Plasmodium yoelii. We observed expression of hPDGFRβ mainly in cells associated with the vasculature suggesting that TRAP:hPDGFRβ interaction may play a role in the recognition of blood vessels by invading sporozoites.

Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.

Summary Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention., Graphical Abstract, Highlights • Specific compounds against P. falciparum Plasmepsin IX and X were identified • PMIX and PMX are modulators of parasite proteins for egress, invasion, and development • Anti-PMIX and anti-PMX compounds inhibit liver, blood, and mosquito stages of Plasmodium • One compound, WM382, can clear mouse models of P. berghei and P. falciparum parasites, We describe inhibitors of essential aspartic proteases from malaria parasites that block multiple life cycle stages. PMIX and PMX are master modulators processing proteins required for invasion, development, and egress. Administration of WM382 cured mice of malaria infection, showing that these inhibitors are promising candidates for malaria treatment and prevention.

Malaria remains a significant public health burden with 214 million new infections and over 400,000 deaths in 2015. Elucidating relevant Plasmodium parasite biology can lead to the identification of novel ways to control and ultimately eliminate the parasite within geographic areas. Particularly, the development of an effective vaccine that targets the clinically silent pre-erythrocytic stages of infection would significantly augment existing malaria elimination tools by preventing both the onset of blood-stage infection/disease as well as spread of the parasite through mosquito transmission. In this Perspective, we discuss the role of small animal models in pre-erythrocytic stage vaccine development, highlighting how human liver-chimeric and human immune system mice are emerging as valuable components of these efforts.

Plasmodium sporozoites infect the liver and develop into exoerythrocytic merozoites that initiate blood-stage disease. The hepatocyte molecular pathways that permit or abrogate parasite replication and merozoite formation have not been thoroughly explored, and a deeper understanding may identify therapeutic strategies to mitigate malaria. Cellular inhibitor of apoptosis (cIAP) proteins regulate cell survival and are co-opted by intracellular pathogens to support development. Here, we show that cIAP1 levels are upregulated during Plasmodium liver infection and that genetic or pharmacological targeting of cIAPs using clinical-stage antagonists preferentially kills infected hepatocytes and promotes immunity. Using gene-targeted mice, the mechanism was defined as TNF-TNFR1-mediated apoptosis via caspases 3 and 8 to clear parasites. This study reveals the importance of cIAPs to Plasmodium infection and demonstrates that host-directed antimalarial drugs can eliminate liver parasites and induce immunity while likely providing a high barrier to resistance in the parasite.

Background Plasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available. Results Here, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite. Conclusions The α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.

Background The assessment of antibody responses after immunization with radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites (Sanaria PfSPZ Vaccine) has focused on IgG isotype antibodies. Here, we aimed to investigate if P. falciparum sporozoite binding and invasion-inhibitory IgM antibodies are induced following immunization of malaria-preexposed volunteers with PfSPZ Vaccine. Methods Using serum from volunteers immunized with PfSPZ, we measured vaccine-induced IgG and IgM antibodies to P. falciparum circumsporozoite protein (PfCSP) via ELISA. Function of this serum as well as IgM antibody fractions was measured via in vitro in an inhibition of sporozoite invasion assay. These IgM antibody fractions were also measured for binding to sporozoites by immunofluorescence assay and complement fixation on whole sporozoites. Results We found that in addition to anti-PfCSP IgG, malaria-preexposed volunteers developed anti-PfCSP IgM antibodies after immunization with PfSPZ Vaccine and that these IgM antibodies inhibited P. falciparum sporozoite invasion of hepatocytes in vitro. These IgM plasma fractions also fixed complement to whole P. falciparum sporozoites. Conclusions This is the first finding that PfCSP and P. falciparum sporozoite-binding IgM antibodies are induced following immunization of PfSPZ Vaccine in malaria-preexposed individuals and that IgM antibodies can inhibit P. falciparum sporozoite invasion into hepatocytes in vitro and fix complement on sporozoites. These findings indicate that the immunological assessment of PfSPZ Vaccine-induced antibody responses could be more sensitive if they include parasite-specific IgM in addition to IgG antibodies. Clinical trials registration NCT02132299.

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete-specific circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) promoter (S4/CelTOSCTRP ), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOSCTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild-type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.

AimsRecent reports on infantile haemangioma (IH) have demonstrated a primitive population of interstitial cells expressing the embryonic transcription factor, Nanog, with decreasing abundance during involution. In this report we investigated the expression of Nanog on mast cells in all three phases of IH progression.MethodsParaffin-embedded sections of six proliferating, six involuting and six involuted IH lesions were used to investigate the expression of tryptase, Nanog, CD45, CD34 and GLUT-1 by immunostaining.ResultsMast cells, identified by their expression of tryptase, were located in the interstitium of IH lesions. 93%, 42% and 0% of these tryptase+ cells also expressed Nanog, in proliferating, involuting and involuted IH, respectively.ConclusionsThe identification of an abundant population of tryptase+/Nanog+ cells in IH is novel. The relative loss of Nanog expression as IH involutes may be a result of maturation and/or proliferation of these cells. This report supports the primitive nature of IH.

Background Verrucous hemangioma (VH) presents clinically as a vascular malformation but has similar histopathologic features to infantile hemangioma. This study characterized the cell population within VH. Material and methods Paraffin-embedded sections from two male patients with VH were processed for immunohistochemistry. The expression of SMA, CD34, glucose transporter-1 (Glut-1), D2-40, brachyury, angiotensin converting enzyme (ACE), Oct-4, hemoglobin ζ chain (HBZ), Wilms tumor protein (WT-1) and CD45 was examined. Results The lymphatic marker, D2-40, was not expressed in VH, whereas Glut-1 was widely expressed in infantile hemangioma, it was only focally expressed by the endothelium of VH. The endothelium of VH expressed the primitive markers, Oct-4, brachyury and ACE. The primitive marker, WT-1, was expressed predominantly on the pericyte layer of both VH and infantile hemangioma. However, HBZ was only expressed in infantile hemangioma. CD45, a mature hematopoetic marker, was expressed by cells within the interstitium, away from the endothelium of VH and infantile hemangioma. Discussion The expression of the primitive markers, Oct-4, brachyury and ACE on the endothelium, and WT-1 predominantly on the pericyte layer of VH shows a primitive microvascular phenotype similar to infantile hemangioma. However, the absence of the embryonic marker, HBZ, expressed only in first trimester placenta and in proliferating infantile hemangioma, suggests a different cellular origin. HBZ could be used to distinguish between the two conditions.

Plasmodium falciparum malaria remains the deadliest parasitic disease worldwide. Vaccines targeting the preerythrocytic sporozoite and liver stages have the potential to entirely prevent blood-stage infection and disease, as well as onward transmission. Sporozoite surface and secreted proteins are leading candidates for inclusion in a preerythrocytic stage-specific, antibody-based vaccine. Preclinical functional assays to identify humoral correlates of protection in vitro and to validate novel sporozoite protein targets for inclusion in multisubunit vaccines currently do not consider the interaction of sporozoite-targeting antibodies with other components of the immune system. Here, we describe the development of a simple flow cytometric assay to quantitatively assess the ability of antibodies directed against P. falciparum sporozoites to facilitate their phagocytosis. We demonstrate that this s porozoite o psonic p hagocytosis a ssay (SOPA) is compatible with both monoclonal antibodies and human immune serum and can be performed using cryopreserved P. falciparum sporozoites. This simple, accessible assay will aid with the assessment of antibody responses to vaccination with Plasmodium antigens and their interaction with phagocytic cells of the immune system.

The origin of infantile haemangioma (IH) remains enigmatic. A primitive mesodermal phenotype origin of IH with the ability to differentiate down erythropoietic and terminal mesenchymal lineages has recently been demonstrated.To investigate the expression of human embryonic stem cell (hESC) markers in IH and to determine whether IH-derived cells have the functional capacity to form teratoma in vivo.Immunohistochemical staining and quantitative reverse transcription PCR were used to investigate the expression of hESC markers in IH biopsies. The ability of cells derived from proliferating IH to form teratomas in a mouse xenograft model was investigated.The hESC markers, Oct-4, STAT-3 and stage-specific embryonic antigen 4 were collectively expressed on the endothelium of proliferating IH lesions, whereas Nanog was not. Nanog was expressed by cells in the interstitium and these cells did not express Oct-4, stage-specific embryonic antigen 4 or STAT-3. Proliferating IH-derived cells were unable to form teratomas in severely compromised immunodeficient/non-obese diabetic mice.The novel expression of hESC on two different populations of cells in proliferating IH and their inability to form teratomas in vivo infer the presence of a primitive cellular origin for IH downstream from hESC.

The antiproliferative effects of opiate exposure on neurogenesis in vitro have been well documented, but the effects of opiates on brain development in vivo are less well understood. We have recently shown that mu opioid receptors are expressed on radial glia of the lateral ventricle, the neuronal and glial progenitor cells of the developing cortex. In the present study we show that in vivo morphine treatment of the E15.5 mouse increases the length of the G(2)/M phase of the radial glial cell cycle in the dorsal telencephalon, as well as slows interkinetic nuclear migration of radial glial nuclei from the basal ventricular zone to the apical surface. A prolonged G(2)/M phase was also observed in basal progenitor cells. Although morphine exposure altered the duration of the cell cycle for progenitor cells in the embryonic telencephalon, it did not affect whether the progenitors remained proliferative and re-entered the S phase, or whether they exited the cell cycle and became quiescent. In addition, morphine treatment did not change the proportion of basal to apical mitoses. These findings indicate that opioid signalling plays a role in cell cycle progression of both radial glia and basal progenitor cells in vivo in the developing cerebral cortex.

Immunization of humans with whole sporozoites confers complete, sterilizing immunity against malaria infection. However, achieving consistent safety while maintaining immunogenicity of whole parasite vaccines remains a formidable challenge. We generated a genetically attenuated Plasmodium falciparum (Pf) malaria parasite by deleting three genes expressed in the pre-erythrocytic stage (Pf p52-/p36-/sap1-). We then tested the safety and immunogenicity of the genetically engineered (Pf GAP3KO) sporozoites in human volunteers. Pf GAP3KO sporozoites were delivered to 10 volunteers using infected mosquito bites with a single exposure consisting of 150 to 200 bites per subject. All subjects remained blood stage-negative and developed inhibitory antibodies to sporozoites. GAP3KO rodent malaria parasites engendered complete, protracted immunity against infectious sporozoite challenge in mice. The results warrant further clinical testing of Pf GAP3KO and its potential development into a vaccine strain.

Summary Propranolol is increasingly used to treat problematic infantile haemangioma (IH), although its molecular mechanisms remain unclear. A key feature of propranolol therapy is the decreased deposition of fibrofatty residuum compared with spontaneously involuting IH. This study investigated the molecular consequences of propranolol treatment for IH in vivo . Immunohistochemical and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining was performed on five age matched patients with proliferative IH. Two patients (A and B) were undergoing propranolol treatment at the time of surgical resection. Propranolol treatment increased apoptosis, and induced mast cells to degranulate and secrete tryptase into the interstitium. The microvessels of patient A were immature [weak von Willibrand Factor (vWF), and strong osteoprotegerin (OPG) staining], comparable to untreated proliferative IH, while those of patient B were mature (strong vWF staining, and no OPG staining). The perivascular CD90 + mesenchymal stem cell population was preserved in both propranolol treated patients. Using rarely obtained biopsies from IH patients treated with propranolol, we show increased apoptosis by propranolol for the first time in vivo . We also suggest that mast cells, through secreted proteases, may contribute to the decreased fibrofatty residuum seen with propranolol treatment.

Cannabis is the most widely used illicit drug, and disruption of learning and memory are commonly reported consequences of cannabis use. We have previously demonstrated a spatial learning impairment by ∆(9)-tetrahydrocannabinol (THC) in adolescent Sprague-Dawley rats (Steel et al., 2011). The molecular mechanisms underlying behavioural impairment by cannabis remain poorly understood, although the importance of adaptive changes in neuroplasticity (synaptic number and strength) and neurogenesis during learning are accepted. Here we aimed to identify any effects of THC on the early induction of these adaptive processes supporting learning, so we conducted our analyses at the mid-training point of our previous study. Both untrained and trained (15 days of training) adolescent (P28-P42) Sprague-Dawley rats were treated daily with THC (6 mg/kg i.p.) or its vehicle, and changes in the levels of markers of hippocampal neuroplasticity (CB1R, PSD95, synapsin-I, synapsin-III) and neurogenesis (Ki67, DCX, PSA-NCAM, BrdU labelling) by training were measured. Training of control animals, but not THC-treated animals increased neuroplasticity marker levels. However training of THC-treated animals, but not control animals reduced immature neuronal marker levels. Levels of hippocampal proliferation, and survival of the BrdU-labelled progeny of these divisions were unaffected by THC in trained and untrained animals. These data show a smaller neuroplastic response, and a reduction of new-born neuronal levels not attributable to effects on proliferation or survival by THC-treatment during training. Importantly no effects of THC were seen in the absence of training, indicating that these effects represent specific impairments by THC on training-induced responses.

Cannabis is the most popular illicit drug used by adolescents. Yet, there are only a few studies that have examined the effects of cannabis use on learning and memory during this sensitive and important neurodevelopmental stage. Male adolescent Sprague-Dawley rats were treated with Δ(9)-tetrahydrocannabinol (THC, 6 mg/kg) daily for 27 days and concurrently trained in a spatial learning and memory task. The chronic effects of cannabis use were specifically examined by assessing animal behaviour during the 'postacute' period (17 h after drug exposure), when minimal acute drug burden is expected to be present. The postacute period is a good model for cannabis use patterns in human adolescents. In addition, we investigated whether the hierarchical organization of working memory (chunking) was impaired by THC-treatment. We show that THC exposure impairs adolescent learning when tested in the postacute period, and that THC impairs the ability of animals to use a chunking strategy.