A new MVA ancestor-derived oncolytic vaccinia virus induces immunogenic tumor cell death and robust antitumor immune responses.

Vaccinia viruses (VACVs) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified VACV Ankara (MVA) is a particularly altered VACV strain that is highly immunogenic, incapable of replicating in mammalian hosts, and broadly used as a safe vector for vaccination. Alternatively, Western Reserve (WR) or Copenhagen (Cop) are VACV strains that efficiently replicate in cancer cells and, therefore, are used to develop oncolytic viruses. However, the immune evasion capacity of WR or Cop hinders their ability to elicit antitumor immune responses, which is crucial for efficacy in the clinic. Here, we describe a new VACV strain named Immune-Oncolytic VACV Ankara (IOVA), which combines efficient replication in cancer cells with induction of immunogenic tumor cell death (ICD). IOVA was engineered from an MVA ancestor and shows superior cytotoxicity in tumor cells. In addition, the IOVA genome incorporates mutations that lead to massive fusogenesis of tumor cells, which contributes to improved antitumor effects. In syngeneic mouse tumor models, the induction of ICD results in robust antitumor immunity directed against tumor neo-epitopes and eradication of large established tumors. These data present IOVA as an improved immunotherapeutic oncolytic vector.

Three dimensional classification of dislocations from single projections.

Many material properties are governed by dislocations and their interactions. The reconstruction of the three-dimensional structure of a dislocation network so far is mainly achieved by tomographic tilt series with high angular ranges, which is experimentally challenging and additionally puts constraints on possible specimen geometries. Here, we show a way to reveal the three dimensional location of dislocations and simultaneously classify their type from single 4D scanning transmission electron microscopy measurements. The dislocation's strain field causes inter-band scattering between the electron's Bloch waves within the crystal. This scattering in turn results in characteristic interference patterns with sufficient information to identify the dislocations type and depth in beam direction by comparison with multi-beam calculations. We expect the presented measurement principle will lead to fully automated methods for reconstruction of the three dimensional strain fields from such measurements with a wide range of applications in material and physical sciences and engineering.

Chronic social defeat alters brain vascular-associated cell gene expression patterns leading to vascular dysfunction and immune system activation.

Brain vascular integrity is critical for brain health, and its disruption is implicated in many brain pathologies, including psychiatric disorders. Brain-vascular barriers are a complex cellular landscape composed of endothelial, glial, mural, and immune cells. Yet currently, little is known about these brain vascular-associated cells (BVACs) in health and disease. Previously, we demonstrated that 14 days of chronic social defeat (CSD), a mouse paradigm that produces anxiety and depressive-like behaviors, causes cerebrovascular damage in the form of scattered microbleeds. Here, we developed a technique to isolate barrier-related cells from the mouse brain and subjected the isolated cells to single-cell RNA sequencing. Using this isolation technique, we found an enrichment in BVAC populations, including distinct subsets of endothelial and microglial cells. In CSD compared to non-stress, home-cage control, differential gene expression patterns disclosed biological pathways involving vascular dysfunction, vascular healing, and immune system activation. Overall, our work demonstrates a unique technique to study BVAC populations from fresh brain tissue and suggests that neurovascular dysfunction is a key driver of psychosocial stress-induced brain pathology.

[Infectious diseases as you have never read them (second part )]. / L'infectiologie comme vous ne l'avez jamais lue (la suite ).

The Covid-19 epidemic has turned our lives upside down; surprisingly certain aspects of this infection can also make you smile, as well as certain modes of transmission of infectious agents that we have grouped together in the form of an original "blooper" of infectious diseases.

L'épidémie de Covid-19 a bouleversé notre vie. Étonnamment, certains aspects de cette infection peuvent aussi faire sourire, tout comme certains modes de transmission d'agents infectieux que nous avons regroupés sous la forme d'un « bêtisier ¼ original des maladies infectieuses.

Navigation with Augmented Reality in a Hospital.

To improve wayfinding in hospitals or other complex buildings a prototype of an indoor navigation app was implemented that uses Augmented Reality for positioning and guiding users. The iOS and Android app allow the navigation from anywhere inside an area of 690 m2. In a usability test 8 from 12 users preferred using the app over a map or verbal directions. Along with the proposed improvements this approach allows covering areas as large as 100'000 m2.

CCR2 monocytes repair cerebrovascular damage caused by chronic social defeat stress.

Immune surveillance of the brain plays an important role in health and disease. Peripheral leukocytes patrol blood-brain barrier interfaces, and after injury, monocytes cross the cerebrovasculature and follow a pattern of pro- and anti-inflammatory activity leading to tissue repair. We have shown that chronic social defeat (CSD) causes scattered vasculature disruptions. Here, we assessed CCR2+ monocyte trafficking to the vascular injury sites in Ccr2wt/rfp reporter mice both during CSD and one week following CSD cessation. We found that CSD for 14 days induced microhemorrhages where plasma fibrinogen leaked into perivascular spaces, but it did not affect the distribution or density of CCR2rfp+ monocytes in the brain. However, after recovery from CSD, many vascularly adhered CCR2+ cells were detected, and gene expression of the CCR2 chemokine receptor ligands CCL7 and CCL12, but not CCL2, was elevated in endothelial cells. Adhered CCR2+ cells were mostly the non-classical, anti-inflammatory Ly6Clo type, and they phagocytosed fibrinogen in perivascular spaces. In CCR2-deficient Ccr2rfp/rfp mice, fibrinogen levels remained elevated in recovery. Fibrinogen infused intracerebroventricularly induced CCR2+ cells to adhere to the vasculature and phagocytose perivascular fibrinogen in Ccr2wt/rfp but not Ccr2rfp/rfp mice. Depletion of monocytes with clodronate liposomes during CSD recovery prevented fibrinogen clearance and blocked behavioral recovery. We hypothesize that peripheral CCR2+ monocytes are not elevated in the brain on day 14 at the end of CSD and do not contribute to its behavioral effects at that time, but in recovery following cessation of stress, they enter the brain and exert restorative functions mediating vascular repair and normalization of behavior.

Mitophagy in the basolateral amygdala mediates increased anxiety induced by aversive social experience.

Psychosocial stress is a common risk factor for anxiety disorders. The cellular mechanism for the anxiogenic effect of psychosocial stress is largely unclear. Here, we show that chronic social defeat (CSD) stress in mice causes mitochondrial impairment, which triggers the PINK1-Parkin mitophagy pathway selectively in the amygdala. This mitophagy elevation causes excessive mitochondrial elimination and consequent mitochondrial deficiency. Mitochondrial deficiency in the basolateral amygdalae (BLA) causes weakening of synaptic transmission in the BLA-BNST (bed nucleus of the stria terminalis) anxiolytic pathway and increased anxiety. The CSD-induced increase in anxiety-like behaviors is abolished in Pink1-/- and Park2-/- mice and alleviated by optogenetic activation of the BLA-BNST synapse. This study identifies an unsuspected role of mitophagy in psychogenetic-stress-induced anxiety elevation and reveals that mitochondrial deficiency is sufficient to increase anxiety and underlies the psychosocial-stress-induced anxiety increase. Mitochondria and mitophagy, therefore, can be potentially targeted to ameliorate anxiety.

A simplified method for bronchoalveolar lavage in mice by orotracheal intubation avoiding tracheotomy.

Bronchoalveolar lavage (BAL) represents an important method to sample immune cells and soluble substances from the lungs of humans and animals suffering from respiratory disease. The mouse is the most commonly used model organism to study lung disease. Performing BAL in mice is difficult due to their small size and the currently used method requires tracheotomy, a complex and time-consuming procedure. Here, we describe a simple alternative procedure that avoids this step. To perform the BAL, a rigid, olive tip cannula is inserted from the mouth into the trachea under visual inspection. This novel method requires minimal training, is simple, fast, inexpensive and should be useful for researchers studying mouse models of human lung disease.

Activation of interferon regulatory factor 3 by replication-competent vaccinia viruses improves antitumor efficacy mediated by T cell responses.

Recently, oncolytic vaccinia viruses (VACVs) have shown their potential to provide for clinically effective cancer treatments. The reason for this clinical usefulness is not only the direct destruction of infected cancer cells but also activation of immune responses directed against tumor antigens. For eliciting a robust antitumor immunity, a dominant T helper 1 (Th1) cell differentiation of the response is preferred, and such polarization can be achieved by activating the Toll-like receptor 3 (TLR3)-interferon regulatory factor 3 (IRF3) signaling pathway. However, current VACVs used as oncolytic viruses to date still encode several immune evasion proteins involved in the inhibition of this signaling pathway. By inactivating genes of selected regulatory virus proteins, we aimed for a candidate virus with increased potency to activate cellular antitumor immunity but at the same time with a fully maintained replicative capacity in cancer cells. The removal of up to three key genes (C10L, N2L, and C6L) from VACV did not reduce the strength of viral replication, both in vitro and in vivo, but resulted in the rescue of IRF3 phosphorylation upon infection of cancer cells. In syngeneic mouse tumor models, this activation translated to enhanced cytotoxic T lymphocyte (CTL) responses directed against tumor-associated antigens and neo-epitopes and improved antitumor activity.

B-cells are abnormal in psychosocial stress and regulate meningeal myeloid cell activation.

There is increasing interest in how immune cells, including those within the meninges at the blood-brain interface, influence brain function and mood disorders, but little data on humoral immunity in this context. Here, we show that in mice exposed to psychosocial stress, there is increased splenic B cell activation and secretion of the immunoregulatory cytokine interleukin (IL)-10. Meningeal B cells were prevalent in homeostasis but substantially decreased following stress, whereas Ly6Chi monocytes increased, and meningeal myeloid cells showed augmented expression of activation markers. Single-cell RNA sequencing of meningeal B cells demonstrated the induction of innate immune transcriptional programmes following stress, including genes encoding antimicrobial peptides that are known to alter myeloid cell activation. Cd19-/- mice, that have reduced B cells, showed baseline meningeal myeloid cell activation and decreased exploratory behaviour. Together, these data suggest that B cells may influence behaviour by regulating meningeal myeloid cell activation.

Diverse roles of phosphatidate phosphatases in insect development and metabolism.

The conversion of the glycerophospholipid phosphatidic acid (PA) into diacylglycerol (DAG) is essential for the biosynthesis of membrane phospholipids and storage fats. Importantly, both PA and DAG can also serve signaling functions in the cell. The dephosphorylation of PA that yields DAG can be executed by two different classes of enzymes, Mg2+-dependent lipins and Mg2+-independent lipid phosphate phosphatases. Here, I will discuss the current status of research directed at understanding the roles of these enzymes in insect development and metabolism. Special emphasis will be given to studies in the model organism Drosophila melanogaster.

Modulation of Cell Surface Receptor Expression by Modified Vaccinia Virus Ankara in Leukocytes of Healthy and HIV-Infected Individuals.

Viral vectors are increasingly used as delivery means to induce a specific immunity in humans and animals. However, they also impact the immune system, and it depends on the given context whether this is beneficial or not. The attenuated vaccinia virus strain modified vaccinia virus Ankara (MVA) has been used as a viral vector in clinical studies intended to treat and prevent cancer and infectious diseases. The adjuvant property of MVA is thought to be due to its capability to stimulate innate immunity. Here, we confirmed that MVA induces interleukin-8 (IL-8), and this chemokine was upregulated significantly more in monocytes and HLA-DRbright dendritic cells (DCs) of HIV-infected patients on combined antiretroviral therapy (ART) than in cells of healthy persons. The effect of MVA on cell surface receptors is mostly unknown. Using mass cytometry profiling, we investigated the expression of 17 cell surface receptors in leukocytes after ex vivo infection of human whole-blood samples with MVA. We found that MVA downregulates most of the characteristic cell surface markers in particular types of leukocytes. In contrast, C-X-C motif chemokine receptor 4 (CXCR4) was significantly upregulated in each leukocyte type of healthy persons. Additionally, we detected a relative higher cell surface expression of the HIV-1 co-receptors C-C motif chemokine receptor 5 (CCR5) and CXCR4 in leukocytes of HIV-ART patients than in healthy persons. Importantly, we showed that MVA infection significantly downregulated CCR5 in CD4+ T cells, CD8+ T cells, B cells, and three different DC populations. CD86, a costimulatory molecule for T cells, was significantly upregulated in HLA-DRbright DCs after MVA infection of whole blood from HIV-ART patients. However, MVA was unable to downregulate cell surface expression of CD11b and CD32 in monocytes and neutrophils of HIV-ART patients to the same extent as in monocytes and neutrophils of healthy persons. In summary, MVA modulates the expression of many different kinds of cell surface receptors in leukocytes, which can vary in cells originating from persons previously infected with other pathogens.

Nuclear translocation ability of Lipin differentially affects gene expression and survival in fed and fasting Drosophila.

Lipins are eukaryotic proteins with functions in lipid synthesis and the homeostatic control of energy balance. They execute these functions by acting as phosphatidate phosphatase enzymes in the cytoplasm and by changing gene expression after translocation into the cell nucleus, in particular under fasting conditions. Here, we asked whether nuclear translocation and the enzymatic activity of Drosophila Lipin serve essential functions and how gene expression changes, under both fed and fasting conditions, when nuclear translocation is impaired. To address these questions, we created a Lipin null mutant, a mutant expressing Lipin lacking a nuclear localization signal (LipinΔNLS ), and a mutant expressing enzymatically dead Lipin. Our data support the conclusion that the enzymatic but not nuclear gene regulatory activity of Lipin is essential for survival. Notably, adult LipinΔNLS flies were not only viable but also exhibited improved life expectancy. In contrast, they were highly susceptible to starvation. Both the improved life expectancy in the fed state and the decreased survival in the fasting state correlated with changes in metabolic gene expression. Moreover, increased life expectancy of fed flies was associated with a decreased metabolic rate. Interestingly, in addition to metabolic genes, genes involved in feeding behavior and the immune response were misregulated in LipinΔNLS flies. Altogether, our data suggest that the nuclear activity of Lipin influences the genomic response to nutrient availability with effects on life expectancy and starvation resistance. Thus, nutritional or therapeutic approaches that aim at lowering nuclear translocation of lipins in humans may be worth exploring.

PET measurement of cyclooxygenase-2 using a novel radioligand: upregulation in primate neuroinflammation and first-in-human study.

BACKGROUND: Cyclooxygenase-2 (COX-2), which is rapidly upregulated by inflammation, is a key enzyme catalyzing the rate-limiting step in the synthesis of several inflammatory prostanoids. Successful positron emission tomography (PET) radioligand imaging of COX-2 in vivo could be a potentially powerful tool for assessing inflammatory response in the brain and periphery. To date, however, the development of PET radioligands for COX-2 has had limited success. METHODS: The novel PET tracer [11C]MC1 was used to examine COX-2 expression [1] in the brains of four rhesus macaques at baseline and after injection of the inflammogen lipopolysaccharide (LPS) into the right putamen, and [2] in the joints of two human participants with rheumatoid arthritis and two healthy individuals. In the primate study, two monkeys had one LPS injection, and two monkeys had a second injection 33 and 44 days, respectively, after the first LPS injection. As a comparator, COX-1 expression was measured using [11C]PS13. RESULTS: COX-2 binding, expressed as the ratio of specific to nondisplaceable uptake (BPND) of [11C]MC1, increased on day 1 post-LPS injection; no such increase in COX-1 expression, measured using [11C]PS13, was observed. The day after the second LPS injection, a brain lesion (~ 0.5 cm in diameter) with high COX-2 density and high BPND (1.8) was observed. Postmortem brain analysis at the gene transcript or protein level confirmed in vivo PET results. An incidental finding in an unrelated monkey found a line of COX-2 positivity along an incision in skull muscle, demonstrating that [11C]MC1 can localize inflammation peripheral to the brain. In patients with rheumatoid arthritis, [11C]MC1 successfully imaged upregulated COX-2 in the arthritic hand and shoulder and apparently in the brain. Uptake was blocked by celecoxib, a COX-2 preferential inhibitor. CONCLUSIONS: Taken together, these results indicate that [11C]MC1 can image and quantify COX-2 upregulation in both monkey brain after LPS-induced neuroinflammation and in human peripheral tissue with inflammation. TRIAL REGISTRATION: ClinicalTrials.gov NCT03912428. Registered April 11, 2019.

Analysis of cerebrovascular dysfunction caused by chronic social defeat in mice.

Psychological stress and affective disorders are clinically associated with hypertension and vascular disease, but the biological links between the conditions have not been fully explored. To examine this relationship, we used chronic social defeat (CSD) stress, which produces anxiety-like and depressive-like behavioral declines in susceptible mice. In such mice, CSD also produces cerebrovascular microbleeds in scattered locations. Here, we showed further evidence of vascular pathology and blood-brain barrier breakdown by visualizing plasma immunoglobulins and erythrocytes within the parenchyma and perivascular spaces of CSD brains. To further characterize the impact of stress on the cerebrovasculature, brain endothelial cells (bECs) were isolated, and global gene expression profiles were generated. Bioinformatic analysis of CSD-induced transcriptional changes in bECs showed enrichment in pathways that delineate the vascular response to injury. These pathways followed a temporal sequence of inflammation, oxidative stress, growth factor signaling, and wound healing (i.e., platelet aggregation, hemostasis, fibrinogen deposition, and angiogenesis). Immunohistochemical staining for markers of fibrinogen deposition and angiogenesis confirmed the existence of the markers at the sites of vascular disruptions. Recovery after CSD cessation was marked by recruitment of leukocytes perhaps participating in vascular repair. The data suggest that co-morbidity of affective disorders and vascular diseases may be attributed in part to a common link in altered endothelial cell function.

Commensal microbiota drive the functional diversification of colon macrophages.

Mononuclear phagocytes are a heterogeneous population of leukocytes essential for immune homeostasis that develop tissue-specific functions due to unique transcriptional programs driven by local microenvironmental cues. Single cell RNA sequencing (scRNA-seq) of colonic myeloid cells from specific pathogen free (SPF) and germ-free (GF) C57BL/6 mice revealed extensive heterogeneity of both colon macrophages (MPs) and dendritic cells (DCs). Modeling of developmental pathways combined with inference of gene regulatory networks indicate two major trajectories from common CCR2+ precursors resulting in colon MP populations with unique transcription factors and downstream target genes. Compared to SPF mice, GF mice had decreased numbers of total colon MPs, as well as selective proportional decreases of two major CD11c+CD206intCD121b+ and CD11c-CD206hiCD121b- colon MP populations, whereas DC numbers and proportions were not different. Importantly, these two major colon MP populations were clearly distinct from other colon MP populations regarding their gene expression profile, localization within the lamina propria (LP) and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations at the molecular level and highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs.

Nef-induced CCL2 Expression Contributes to HIV/SIV Brain Invasion and Neuronal Dysfunction.

C-C motif chemokine ligand 2 (CCL2) is a chemoattractant for leukocytes including monocytes, T cells, and natural killer cells and it plays an important role in maintaining the integrity and function of the brain. However, there is accumulating evidence that many neurological diseases are attributable to a dysregulation of CCL2 expression. Acquired immune deficiency syndrome (AIDS) encephalopathy is a severe and frequent complication in individuals infected with the human immunodeficiency virus (HIV) or the simian immunodeficiency virus (SIV). The HIV and SIV Nef protein, a progression factor in AIDS pathology, can be transferred by microvesicles including exosomes and tunneling nanotubes (TNT) within the host even to uninfected cells, and Nef can induce CCL2 expression. This review focuses on findings which collectively add new insights on how Nef-induced CCL2 expression contributes to neurotropism and neurovirulence of HIV and SIV and elucidates why adjuvant targeting of CCL2 could be a therapeutic option for HIV-infected persons.

Nanosecond electron holography by interference gating.

The interference gating is a novel method for robust time-resolved electron holographic measurements by directly switching the interference. Here, a new arrangement is presented in which a biprism in the condenser aperture as a fast electric phase shifter is used to control the interference pattern. High-frequency stimulation of the electric phase shifter in the gigahertz range are performed and observed via electron holography, proving the feasibility of interference gating in the upper picosecond range. Despite the bandwidth limitation of 180 MHz of the current signal generator, a time resolution of 100 ns is achieved through forward correction of the control signal. With this time resolution, it is already possible to measure the transient response of the biasing holder system. Our method paves the way towards a closer look on fast dynamic processes with high temporal and spatial resolution.

The Behavioral Sequelae of Social Defeat Require Microglia and Are Driven by Oxidative Stress in Mice.

Chronic social defeat (CSD) in male mice can produce anxiety and aberrant socialization. Animals susceptible to CSD show activation of microglia, which have elevated levels of oxidative stress markers. We hypothesized that microglia and reactive oxygen species (ROS) production contribute to the CSD stress-induced changes in affective behavior. First, we selectively depleted microglia (99%) by administering the CSF1R (colony-stimulating factor 1 receptor) antagonist PLX5622 before and during the 14 d CSD procedure. Microglia-depleted mice in contrast to nondepleted mice were protected from the stress effects measured by light/dark and social interaction tests. ROS production, measured histochemically following dihydroethidium administration, was elevated by CSD, and the production was reduced to basal levels in mice lacking microglia. The deleterious stress effects were also blocked in nondepleted mice by continuous intracerebral administration of N-acetylcysteine (NAC), a ROS inhibitor. In a second experiment, at the end of the CSD period, PLX5622 was discontinued to allow microglial repopulation. After 14 d, the brain had a full complement of newly generated microglia. At this time, the mice that had previously been protected now showed behavioral deficits, and their brain ROS production was elevated, both in all brain cells and in repopulated microglia. NAC administration during repopulation prevented the behavioral decline in the repopulated mice, and it supported behavioral recovery in nondepleted stressed mice. The data suggest that microglia drive elevated ROS production during and after stress exposure. This elevated ROS activity generates a central state supporting dysregulated affect, and it hinders the restoration of behavioral and neurochemical homeostasis after stress cessation.SIGNIFICANCE STATEMENT Chronic psychosocial stress is associated with psychiatric disorders such as depression and anxiety. Understanding the details of CNS cellular contributions to stress effects could lead to the development of intervention strategies. Inflammation and oxidative stress are positively linked to depression severity, but the cellular nature of these processes is not clear. The chronic social defeat (CSD) paradigm in mice produces mood alterations and microglial activation characterized by elevated reactive oxygen species (ROS) production. The depletion of microglia or ROS inhibition prevented adverse stress effects. Microglial repopulation of the brain post-CSD reintroduced adverse stress effects, and ROS inhibition in this phase protected against the effects. The results suggest that stress-induced microglial ROS production drives a central state that supports dysregulated affective behavior.