In situ fate of Chikungunya virus replication organelles.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.

Characterization of macrophages, giant cells and granulomas during muscle regeneration after irradiation.

Macrophages play a fundamental role in the different stages of muscle regeneration although the precise mechanisms involved are not entirely understood. Here we investigated the types of macrophages and cytokines that appeared in muscles after local gamma irradiation of mini-pigs that underwent no subsequent treatment or received three successive adipose tissue-derived stem cell (ASC) injections. Although some variability was observed among the three animals included in each study group, a general picture emerged. No macrophages appeared in control muscles from regions that had not been irradiated nor in muscles from irradiated regions derived from two animals. A third irradiated, but untreated animal, with characteristic muscle fibrosis and necrosis due to irradiation, showed invasion of M2 macrophages within small muscle lesions. In contrast, among the three ASC-treated and irradiated animals, one of them had completely recovered normal muscle architecture at the time of sampling with no invading macrophages, muscle from a second one contained mostly M1 macrophages and some M2-like macrophages whereas muscle from a third one displayed granulomas and giant cells. ASC treatment was associated with the presence of similar levels of pro-inflammatory cytokines within the two animals in the process of muscle regeneration whereas the levels of IL-4 and IL-10 expression were distinct from one animal to another. Microspectrofluorimetry and in situ hybridization revealed strong expression of TGF-ß1 and TNFα in regenerating muscle. Overall, the data confirm the critical role of macrophages in muscle regeneration and suggest the involvement of a complex network of cytokine expression for successful recovery.

Hybridization-chain-reaction is a relevant method for in situ detection of M2d-like macrophages in a mini-pig model.

Macrophages are a heterogeneous population of cells with an important role in innate immunity and tissue regeneration. Based on in vitro experiments, macrophages have been subdivided into five distinct subtypes named M1, M2a, M2b, M2c, and M2d, depending on the means of their activation and the cell surface markers they display. Whether all subtypes can be detected in vivo is still unclear. The identification of macrophages in vivo in the regenerating muscle could be used as a new diagnostic tool to monitor therapeutic strategies for tissue repair. The use of classical immunolabeling techniques is unable to discriminate between different M2 macrophages and a functional characterization of these macrophages is lacking. Using in situ hybridization coupled with hybridization-chain-reaction detection (HCR), we achieved the identification of M2d-like macrophages within regenerating muscle and applied this technique to understand the role of M2 macrophages in the regeneration of irradiated pig-muscle after adipose tissue stem cell treatment. Our work highlights the limits of immunolabeling and the usefulness of HCR analysis to provide valuable information for macrophage characterization.

Nose-only inhalations of high-dose alumina nanoparticles/hydrogen chloride gas mixtures induce strong pulmonary pro-inflammatory response: a pilot study.

OBJECTIVE: Solid composite propellants combustion, in aerospace and defense fields, can lead to complex aerosols emission containing high concentrations of alumina nanoparticles (Al2O3 NPs) and hydrogen chloride gas (HClg). Exposure to these mixtures by inhalation is thus possible but literature data toward their pulmonary toxicity are missing. To specify hazards resulting from these combustion aerosols, a pilot study was implemented. MATERIALS AND METHODS: Male Wistar rats were nose-only exposed to Al2O3 NPs (primary size 13 nm, 10 g/L suspension leading to 20.0-22.1 mg/m3 aerosol) and/or to HClg aerosols (5 ppm target concentration) following two exposure scenarios (single exposures (SE) or repeated exposures (RE)). Bronchoalveolar lavage fluids (BALF) content and lungs histopathology were analyzed 24 h after exposures. RESULTS: Repeated co-exposures increased total proteins and LDH concentrations in BALF indicating alveolar-capillary barrier permeabilization and cytolysis. Early pulmonary inflammation was induced after RE to Al2O3 NPs ± HClg resulting in PMN, TNF-α, IL-1ß, and GRO/KC increases in BALF. Both exposure scenarios resulted in pulmonary histopathological lesions (vascular congestions, bronchial pre-exfoliations, vascular and interalveolar septum edemas). Lung oxidative damages were observed in situ following SE. CONCLUSION: Observed biological effects are dependent on both aerosol content and exposure scenario. Results showed an important pro-inflammatory effect of Al2O3 NPs/HClg mixtures on the lungs of rat 24 h after exposure. This pilot study raises concerns toward potential long-term pulmonary toxicity of combustion aerosols and highlights the importance for further studies to be led in order to define dose limitations and exposure thresholds for risk management at the work place.

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein.

UNLABELLED: Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. IMPORTANCE: A specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the balance toward its elimination. An interaction between the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of Ebola virus GP occurred. In this model, the enhancement of infection was shown to be independent of the serum complement. The facilitation of EBOV entry into target host cells by the interaction with ficolin-1 and other host lectins shunts virus elimination, which likely facilitates the survival of the virus in infected host cells and contributes to the virus strategy to subvert the innate immune response.

Parapoxviruses, little-known zoonotic pathogens.

Parapoxviruses, double-stranded DNA viruses of the Poxviridæ family, are etiologic agents of cutaneaous infectious diseases among farm animals. These highly contagious viruses are responsible for wide outbreaks among livestock. The clinical manifestations are generally mild and consist of cutaneous or mucosal lesions, which resolve spontaneously within a few weeks. However, secondary bacterial or fungal infections on the lesion sites can aggravate the symptoms. Sore lesions located within the oral cavity and on the udders can impair feeding or nursing, thus leading to death. Livestock parapoxviruses can infect humans by direct or indirect transmission and affect mainly farmers, slaughters and veterinarians. Human symptoms generally consist of small cutaneous lesions located at the inoculation points but more severe forms can occur, peculiarly in immunocompromised persons. The parapoxvirus epidemiology is poorly understood: their respective host range and ecology among wild animals are to be clarified. The identification of parapoxviruses among marine mammals suggests that the genetic diversity within the genus is still underestimated.

Comparison of Three Antagonists of Hedgehog Pathway to Promote Skeletal Muscle Regeneration after High Dose Irradiation.

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept.

Concomitant human infections with 2 cowpox virus strains in related cases, France, 2011.

We investigated 4 related human cases of cowpox virus infection reported in France during 2011. Three patients were infected by the same strain, probably transmitted by imported pet rats, and the fourth patient was infected by another strain. The 2 strains were genetically related to viruses previously isolated from humans with cowpox infection in Europe.

Molecular and Cellular Mechanisms of Inflammation and Tissue Regeneration.

Over the past 70 years, significant progress has been made in understanding the molecular and cellular mechanisms of inflammation and tissue regeneration [...].

In Situ Identification of Both IL-4 and IL-10 Cytokine-Receptor Interactions during Tissue Regeneration.

Cytokines secreted by individual immune cells regulate tissue regeneration and allow communication between various cell types. Cytokines bind to cognate receptors and trigger the healing process. Determining the orchestration of cytokine interactions with their receptors on their cellular targets is essential to fully understanding the process of inflammation and tissue regeneration. To this end, we have investigated the interactions of Interleukin-4 cytokine (IL-4)/Interleukin-4 cytokine receptor (IL-4R) and Interleukin-10 cytokine (IL-10)/Interleukin-10 cytokine receptor (IL-10R) using in situ Proximity Ligation Assays in a regenerative model of skin, muscle and lung tissues in the mini-pig. The pattern of protein-protein interactions was distinct for the two cytokines. IL-4 bound predominantly to receptors on macrophages and endothelial cells around the blood vessels while the target cells of IL-10 were mainly receptors on muscle cells. Our results show that in situ studies of cytokine-receptor interactions can unravel the fine details of the mechanism of action of cytokines.

Deletion of major nonessential genomic regions in the vaccinia virus Lister strain enhances attenuation without altering vaccine efficacy in mice.

The vaccinia virus (VACV) Lister strain was one of the vaccine strains that enabled smallpox eradication. Although the strain is most often harmless, there have been numerous incidents of mild to life-threatening accidents with this strain and others. In an attempt to further attenuate the Lister strain, we investigated the role of 5 genomic regions known to be deleted in the modified VACV Ankara (MVA) genome in virulence in immunodeficient mice, immunogenicity in immunocompetent mice, and vaccine efficacy in a cowpox virus challenge model. Lister mutants were constructed so as to delete each of the 5 regions or various combinations of these regions. All of the mutants replicated efficiently in tissue culture except region I mutants, which multiplied more poorly in human cells than the parental strain. Mutants with single deletions were not attenuated or only moderately so in athymic nude mice. Mutants with multiple deletions were more highly attenuated than those with single deletions. Deleting regions II, III, and V together resulted in total attenuation for nude mice and partial attenuation for SCID mice. In immunocompetent mice, the Lister deletion mutants induced VACV specific humoral responses equivalent to those of the parental strain but in some cases lower cell-mediated immune responses. All of the highly attenuated mutants protected mice from a severe cowpox virus challenge at low vaccine doses. The data suggest that several of the Lister mutants combining multiple deletions could be used in smallpox vaccination or as live virus vectors at doses equivalent to those used for the traditional vaccine while displaying increased safety.

Role of sulfatide in vaccinia virus infection.

BACKGROUND INFORMATION: Vaccinia virus (VACV) was used as a surrogate of variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection. VACV infects cells via attachment and fusion of the viral membrane with the host cell membrane. Glycosphingolipids, expressed in multiple organs, are major components of lipid rafts and have been associated with the infectious route of several pathogens. RESULTS: We demonstrate that the VACV-WR (VACV Western-Reserve strain) displays no binding to Cer (ceramide) or to Gal-Cer (galactosylceramide), but binds to a natural sulfated derivative of these molecules: the Sulf (sulfatide) 3' sulfogalactosylceramide. The interaction between Sulf and VACV-WR resulted in a time-dependent inhibition of virus infection. Virus cell attachment was the crucial step inhibited by Sulf. Electron microscopy showed that SUVs (small unilamellar vesicles) enriched in Sulf bound to VACV particles. Both the A27 and L5 viral membrane proteins were shown to interact with Sulf, indicating that they could be the major viral ligands for Sulf. Soluble Sulf was successful in preventing mortality, but not morbidity, in a lethal mouse model infection with VACV-WR. CONCLUSIONS: Together the results suggest that Sulf could play a role as an alternate receptor for VACV-WR and probably other Orthopoxviruses.

In Situ Gene Expression in Native Cryofixed Bone Tissue.

Bone is a very complex tissue that is constantly changing throughout the lifespan. The precise mechanism of bone regeneration remains poorly understood. Large bone defects can be caused by gunshot injury, trauma, accidents, congenital anomalies and tissue resection due to cancer. Therefore, understanding bone homeostasis and regeneration has considerable clinical and scientific importance in the development of bone therapy. Macrophages are well known innate immune cells secreting different combinations of cytokines and their role in bone regeneration during bone healing is essential. Here, we present a method to identify mRNA transcripts in cryosections of non-decalcified rat bone using in situ hybridization and hybridization chain reaction to explore gene expression in situ for better understanding the gene expression of the bone tissues.

Macrophages Characterization in an Injured Bone Tissue.

Biomaterial use is a promising approach to facilitate wound healing of the bone tissue. Biomaterials induce the formation of membrane capsules and the recruitment of different types of macrophages. Macrophages are immune cells that produce diverse combinations of cytokines playing an important role in bone healing and regeneration, but the exact mechanism remains to be studied. Our work aimed to identify in vivo macrophages in the Masquelet induced membrane in a rat model. Most of the macrophages in the damaged area were M2-like, with smaller numbers of M1-like macrophages. In addition, high expression of IL-1ß and IL-6 cytokines were detected in the membrane region by RT-qPCR. Using an innovative combination of two hybridization techniques (in situ hybridization and in situ hybridization chain reaction (in situ HCR)), M2b-like macrophages were identified for the first time in cryosections of non-decalcified bone. Our work has also demonstrated that microspectroscopical analysis is essential for macrophage characterization, as it allows the discrimination of fluorescence and autofluorescence. Finally, this work has revealed the limitations of immunolabelling and the potential of in situ HCR to provide valuable information for in vivo characterization of macrophages.

Precision Phenotyping of Nectar-Related Traits Using X-ray Micro Computed Tomography.

Flower morphologies shape the accessibility to nectar and pollen, two major traits that determine plant-pollinator interactions and reproductive success. Melon is an economically important crop whose reproduction is completely pollinator-dependent and, as such, is a valuable model for studying crop-ecological functions. High-resolution imaging techniques, such as micro-computed tomography (micro-CT), have recently become popular for phenotyping in plant science. Here, we implemented micro-CT to study floral morphology and honey bees in the context of nectar-related traits without a sample preparation to improve the phenotyping precision and quality. We generated high-quality 3D models of melon male and female flowers and compared the geometric measures. Micro-CT allowed for a relatively easy and rapid generation of 3D volumetric data on nectar, nectary, flower, and honey bee body sizes. A comparative analysis of male and female flowers showed a strong positive correlation between the nectar gland volume and the volume of the secreted nectar. We modeled the nectar level inside the flower and reconstructed a 3D model of the accessibility by honey bees. By combining data on flower morphology, the honey bee size and nectar volume, this protocol can be used to assess the flower accessibility to pollinators in a high resolution, and can readily carry out genotypes comparative analysis to identify nectar-pollination-related traits.

Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

Background: Radiation cystitis (RC) results from chronic inflammation, fibrosis, and vascular damage. The urinary symptoms it causes have a serious impact on patients' quality of life. Despite the improvement in irradiation techniques, the incidence of radiation cystitis remains stable over time, and the therapeutic possibilities remain limited. Mesenchymal stem/stromal cells (MSC) appear to offer2 a promising therapeutic approach by promoting tissue repair through their paracrine action via extracellular vesicles (MSC-EVs) or conditioned medium from human mesenchymal stromal cells (MSC-CM). We assess the therapeutic potential of MSC-EVs or MSC-CM in an in vitro model of RC. Methods:in vitro RC was induced by irradiation of human bladder fibroblasts (HUBF) with the small-animal radiation research platform (SARRP). HUBF were induced towards an RC phenotype after 3 × 3.5 Gy irradiation in the presence of either MSC-EVs or MSC-CM, to assess their effect on fibrosis, angiogenesis, and inflammatory markers. Results: Our data revealed in vitro a higher therapeutic potential of MSC-EVs and MSC-CM in prevention of RC. This was confirmed by down-regulation of α-SMA and CTGF transcription, and the induction of the secretion of anti-fibrotic cytokines, such as IFNγ, IL10 and IL27 and the decrease in the secretion of pro-fibrotic cytokines, IGFBP2, IL1ß, IL6, IL18, PDGF, TNFα, and HGF, by irradiated HUBFs, conditioned with MSC-EVs or MSC-CM. The secretome of MSC (MSC-CM) or its subsecretome (MSC-EVs) are proangiogenic, with the ability to induce vessels from HUVEC cells, ensuring the management of bladder vascular lesions induced by irradiation. Conclusion: MSC-EVs and MSC-CM appear to have promising therapeutic potential in the prevention of RC in vitro, by targeting the three main stages of RC: fibrosis, inflammation and vascular damage.

Influence of the Immune Microenvironment Provided by Implanted Biomaterials on the Biological Properties of Masquelet-Induced Membranes in Rats: Metakaolin as an Alternative Spacer.

Macrophages play a key role in the inflammatory phase of wound repair and foreign body reactions-two important processes in the Masquelet-induced membrane technique for extremity reconstruction. The macrophage response depends largely on the nature of the biomaterials implanted. However, little is known about the influence of the macrophage microenvironment on the osteogenic properties of the induced membrane or subsequent bone regeneration. We used metakaolin, an immunogenic material, as an alternative spacer to standard polymethylmethacrylate (PMMA) in a Masquelet model in rats. Four weeks after implantation, the PMMA- and metakaolin-induced membranes were harvested, and their osteogenic properties and macrophage microenvironments were investigated by histology, immunohistochemistry, mass spectroscopy and gene expression analysis. The metakaolin spacer induced membranes with higher levels of two potent pro-osteogenic factors, transforming growth factor-ß (TGF-ß) and bone morphogenic protein-2 (BMP-2). These alternative membranes thus had greater osteogenic activity, which was accompanied by a significant expansion of the total macrophage population, including both the M1-like and M2-like subtypes. Microcomputed tomographic analysis showed that metakaolin-induced membranes supported bone regeneration more effectively than PMMA-induced membranes through better callus properties (+58%), although this difference was not significant. This study provides the first evidence of the influence of the immune microenvironment on the osteogenic properties of the induced membranes.

Tecovirimat is effective against human monkeypox virus in vitro at nanomolar concentrations.

The ongoing monkeypox virus (MPXV) outbreak is the largest ever recorded outside of Africa. We isolated and sequenced a virus from the first clinical MPXV case diagnosed in France (May 2022). We report that tecovirimat (ST-246), a US Food and Drug Administration approved drug, is efficacious against this isolate in vitro at nanomolar concentrations, whereas cidofovir is only effective at micromolar concentrations. Our results support the use of tecovirimat in ongoing human clinical trials.

Macrophage Identification In Situ.

Understanding the processes of inflammation and tissue regeneration after injury is of great importance. For a long time, macrophages have been known to play a central role during different stages of inflammation and tissue regeneration. However, the molecular and cellular mechanisms by which they exert their effects are as yet mostly unknown. While in vitro macrophages have been characterized, recent progress in macrophage biology studies revealed that macrophages in vivo exhibited distinctive features. Actually, the precise characterization of the macrophages in vivo is essential to develop new healing treatments and can be approached via in situ analyses. Nowadays, the characterization of macrophages in situ has improved significantly using antigen surface markers and cytokine secretion identification resulting in specific patterns. This review aims for a comprehensive overview of different tools used for in situ macrophage identification, reporter genes, immunolabeling and in situ hybridization, discussing their advantages and limitations.

Non-Specific Binding, a Limitation of the Immunofluorescence Method to Study Macrophages In Situ.

Advances in understanding tissue regenerative mechanisms require the characterization of in vivo macrophages as those play a fundamental role in this process. This characterization can be approached using the immuno-fluorescence method with widely studied and used pan-markers such as CD206 protein. This work investigated CD206 expression in an irradiated-muscle pig model using three different antibodies. Surprisingly, the expression pattern during immunodetection differed depending on the antibody origin and could give some false results. False results are rarely described in the literature, but this information is essential for scientists who need to characterize macrophages. In this context, we showed that in situ hybridization coupled with hybridization-chain-reaction detection (HCR) is an excellent alternative method to detect macrophages in situ.