The diffusion of normal skin wound myofibroblast-derived microvesicles differs according to matrix composition.

Microvesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information over long distances, affecting normal and pathological processes including skin wound healing. However, the diffusion of MVs into tissues can be impeded by the extracellular matrix (ECM). We investigated the diffusion of dermal wound myofibroblast-derived MVs into the ECM by using hydrogels composed of different ECM molecules such as fibrin, type III collagen and type I collagen that are present during the healing process. Fluorescent MVs mixed with hydrogels were employed to detect MV diffusion using fluorometric methods. Our results showed that MVs specifically bound type I collagen and diffused freely out of fibrin and type III collagen. Further analysis using flow cytometry and specific inhibitors revealed that MVs bind to type I collagen via the α2ß1 integrin. These data demonstrate that MV transport depends on the composition of the wound environment.

Microvesicles derived from dermal myofibroblasts modify the integrity of the blood and lymphatic barriers using distinct endocytosis pathways.

Microvesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information from their producer cells to target cells. This communication can in turn affect both normal and pathological processes. Mounting evidence has revealed that dermal wound myofibroblasts (Wmyo) produce MVs, which can transfer biomolecules impacting receptor cells such as human dermal microvascular endothelial cells (HDMECs). While the effects of MVs on HDMECs are generally well described in the literature, little is known about the transport of MVs across the HDMEC barrier, and their potential effect on the barrier integrity remains unknown. Here, we investigated these roles of Wmyo-derived MVs on two sub-populations of HDMECs, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs). Using an in vitro model to mimic the endothelial barrier, we showed that MVs crossed the LEC barrier but not the BEC barrier. In addition, we demonstrated that MVs were able to influence the cell-cell junctions of HDMECs. Specifically, we observed that after internalization via the predominantly caveolin-dependent pathway, MVs induced the opening of junctions in BECs. Conversely, in LECs, MVs mainly use the macropinocytosis pathway and induce closure of these junctions. Moreover, proteins in the MV membrane were responsible for this effect, but not specifically those belonging to the VEGF family. Finally, we found that once the LEC barrier permeability was reduced by MV stimuli, MVs ceased to cross the barrier. Conversely, when the BEC barrier was rendered permeable following stimulation with MVs, they were subsequently able to cross the barrier via the paracellular pathway. Taken together, these results suggest that the study of Wmyo-derived MVs offers valuable insights into their interaction with the HDMEC barrier in the context of wound healing. They highlight the potential significance of these MVs in the overall process.

Antimicrobial susceptibility of mastitis pathogens isolated from North American dairy cattle, 2011-2022.

A total of 10,890 bacterial isolates of Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Escherichia coli isolated as etiological agents from dairy cows with mastitis by 29 veterinary laboratories across North America between 2011 and 2022 were tested for in vitro antimicrobial susceptibility by broth microdilution to ampicillin, cefoperazone, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin-novobiocin and pirlimycin according to CLSI standards. Using available clinical breakpoints, antimicrobial resistance among S. dysgalactiae (n = 2406) was low for penicillin-novobiocin (0% resistance), ceftiofur (0.1%), erythromycin (3.2%) and pirlimycin (4.6%). Among S. uberis (n = 2398), resistance was low for ampicillin (0%) and ceftiofur (0.2%) and moderate for erythromycin (11.9%) and pirlimycin (18.4%). For S. aureus (n = 3194), resistance was low for penicillin-novobiocin (0%), ceftiofur (0.1%), oxacillin (0.2%), erythromycin (0.7%), cefoperazone (1.2%) and pirlimycin (2.8%). For E. coli (n = 2892), resistance was low for ceftiofur (2.8%) and cefoperazone (3.4%) and moderate for ampicillin (9.2%). Overall, the results indicate that mastitis pathogens in the United States and Canada have not shown any substantial changes in the in vitro susceptibility to antimicrobial drugs over the 12 years of the study, or among that of the proceeding survey from 2002-2010. The data support the conclusion that resistance to common antimicrobial drugs among mastitis pathogens, even to drugs that have been used in dairies for mastitis management for many years, continues to remain low.

Extracellular vesicles on the move: Traversing the complex matrix of tissues.

Extracellular vesicles are small particles involved in intercellular signaling. They are produced by virtually all cell types, transport biological molecules, and are released into the extracellular space. Studies on extracellular vesicles have become more numerous in recent years, leading to promising research on their potential impact on health and disease. Despite significant progress in understanding the bioactivity of extracellular vesicles, most in vitro and in vivo studies overlook their transport through the extracellular matrix in tissues. The interaction or free diffusion of extracellular vesicles in their environment can provide valuable insights into their efficacy and function. Therefore, understanding the factors that influence the transport of extracellular vesicles in the extracellular matrix is essential for the development of new therapeutic approaches that involve the use of these extracellular vesicles. This review discusses the importance of the interaction between extracellular vesicles and the extracellular matrix and the different factors that influence their diffusion. In addition, we evaluate their role in tissue homeostasis, pathophysiology, and potential clinical applications. Understanding the complex interaction between extracellular vesicles and the extracellular matrix is critical in order to develop effective strategies to target specific cells and tissues in a wide range of clinical applications.

Prevalence and risk factors of toxigenic Clostridioides difficile asymptomatic carriage in 11 French hospitals.

Clostridioides difficile infection (CDI) incidence has increased over the last 20 years. Studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We conducted a point prevalence study to estimate the toxigenic C. difficile asymptomatic carriage rate and the associated risk factors in patients >3 years old. Between September 16, 2019 and January 15, 2020, all patients hospitalized in 11 healthcare facilities in the Paris urban area were included in the study. They were screened on the day of the survey for toxigenic C. difficile carriage by rectal swab and interviewed. Isolates were characterized by PCR ribotyping and multiplex PCR targeting toxin genes. A logistic regression model was used to determine the risk factors associated with toxigenic C. difficile asymptomatic carriage using uni- and multivariate analysis in the subpopulation of patients >3 years old. During the study period, 2,389 patients were included and screened. The median age was 62 years (interquartile range 35-78 years) and 1,153 were male (48.3%). Nineteen patients had a previous CDI (0.9%). Overall, 185/2389 patients were positive for C. difficile (7.7%), including 93 toxigenic strains (3.9%): 77 (82.8%) were asymptomatic (prevalence 3.2%) whereas 12 (12.9%) were diarrheic. Prevalences of toxigenic C. difficile were 3.5% in patients >3 years old and 7.0% in ≤3 years old subjects, respectively. Toxigenic strains mainly belonged to PCR ribotypes 106 (n = 14, 15.0%), 014 (n = 12, 12.9%), and 020 (n = 10, 10.8%). Among toxigenic strains, 6 (6.4%) produced the binary toxin. In multivariate analysis, two factors were positively associated with toxigenic C. difficile asymptomatic carriage in patients >3 years old: multidrug-resistant organisms co-carriage [adjusted Odd Ratio (aOR) 2.3, CI 95% 1.2-4.7, p = 0.02] and previous CDI (aOR 5.8, CI 95% 1.2-28.6, p = 0.03). Conversely, consumption of raw milk products were associated with reduced risk of toxigenic C. difficile colonization (aOR 0.5, CI 95% 0.2-0.9, p = 0.01). We showed that there was a low prevalence of asymptomatic toxigenic C. difficile carriage in hospitalized patients. Consumption of raw milk prevents toxigenic C. difficile colonization, probably due to the barrier effect of milk-associated bacteria.

An in vitro autologous, vascularized, and immunocompetent Tissue Engineered Skin model obtained by the self-assembled approach.

A complete in vitro skin model, containing resident cell types is needed to understand physiology and to consider the role of immune and endothelial cells in dermal drug testing. In this study, a cell extraction technique was developed to isolate resident skin cells from the same human donor while preserving the immune and endothelial cells. Then those cells were used to reconstruct an autologous, vascularized, and immunocompetent Tissue-Engineered Skin model, aviTES. Phenotypic characterization of the viable cells was performed on freshly isolated cells and after thawing through flow cytometry. Dermal cell extracts were characterized as fibroblasts, endothelial and immune cells, and the average amount of each cell type represents 4, 0.5, and 1 million viable cells per g of the dermis, respectively. The 3D models, TES and aviTES, were characterized by a fully differentiated epidermis that showed an increase in the presence of Ki67+ cells in the basolateral layer of the aviTES model. Capillary-like network formation, through the self-assembly of endothelial cells, and the presence of functional immune cells were identified through immunofluorescence staining in aviTES. In addition, the aviTES model was immunocompetent, as evidenced by its capacity to increase the production of pro-inflammatory cytokines TNF-α, MIP-1α, and GM-CSF following LPS stimulation. This study describes an autologous skin model containing a functional resident skin immune system and a capillary network. It provides a relevant tool to study the contribution of the immune system to skin diseases and inflammatory responses and to investigate resident skin cell interactions and drug development. STATEMENT OF SIGNIFICANCE: There is an urgent need for a complete in vitro skin model containing the resident cell types to better understand the role of immune and endothelial cells in skin and to be able to use it for drug testing. Actual 3D models of human skin most often contain only fibroblasts and keratinocytes with a limited number of models containing endothelial cells or a limited variety of immune cells. This study describes an autologous skin model containing a functional resident skin immune system and a capillary network. It provides a relevant tool to study the contribution of the immune system to skin diseases and inflammatory responses and to investigate interactions between resident skin cell, improving our capacity to develop new drugs.

UTP increases wound healing in the self assembled skin substitute (SASS).

The therapeutic potential of purinergic signaling has been explored for a wide variety of diseases, including those related to the skin. In this study, we used the self-assembled skin substitutes (SASS), a highly functional reconstructed human skin model, which shares many properties with normal human skin, to study the impact of purinergic receptors agonists, such as ATP, UTP and a P2Y receptor antagonist, Reactive Blue 2 during wound healing. After treating the wounded skins, we evaluated the wound area, reepithelialization, length of migrating tongues toward the wound, quality of the skins through the cytokeratin 10 and laminin-5 expression, epidermal and dermal cell proliferation. In addition, the expression of the main ectoenzymes capable of hydrolyzing nucleotides were investigated through the wounded SASS regions: unwounded region, wound margin, intermediate region and migrating epidermal tongue. After 3 days, under the UTP treatment, the wounded SASS showed an increase in the reepithelialization and in the proliferation of keratinocytes and fibroblasts, without altering the quality of the skin. We also identified the presence of the ectoenzymes NTPDase1 and NPP1 in the reconstructed human skin model, suggesting their involvement in wound healing. Considering the need for new therapies capable of promoting healing in complex wounds, although these results are still preliminary, they suggest the involvement of extracellular nucleotides in human skin healing and the importance to understand their role in this mechanism. New experiments it will be necessary to determine the mechanisms by which the purinergic signaling is involved in the skin wound healing.

The Self-Assembled Skin Substitute History: Successes, Challenges, and Current Treatment Indications.

The self-assembled skin substitute (SASS) is an autologous bilayered skin substitute designed by our academic laboratory, the Laboratoire d'Organogenèse Expérimentale (LOEX) to offer definitive treatment for patients lacking donor sites (unwounded skin) to cover their burn wounds. This product shows skin-like attributes, such as an autologous dermal and epidermal layer, and is easily manipulable by the surgeon. Its development stems from the need for skin replacement in high total body surface area burned survivors presenting few donor sites for standard split-thickness skin grafting. This review aims to present the history, successes, challenges, and current therapeutic indications of this skin substitute. We review the product's development history, before discussing current production techniques, as well as clinical use. The progression observed since the initial SASS production technique described in 1999, up to the most recent technique expresses significant advances made in the technical aspect of our product, such as the reduction of the production time. We then explore the efficacy and benefits of SASS over existing skin substitutes and discuss the outcomes of a recent study focusing on the successful treatment of 14 patients. Moreover, an ongoing cross-Canada study is further assessing the product's safety and efficacy. The limitations and technical challenges of SASS are also discussed.

Photoprotective effect of solid lipid nanoparticles of rutin against UVB radiation damage on skin biopsies and tissue-engineered skin.

Solid lipid nanoparticles (SLNs) containing rutin were prepared to enhance their photochemopreventive effect on the skin. SLNs were produced by the hot melt microemulsion technique. Two 3D skin models: ex vivo skin explants and 3D tissue engineering skin were used to evaluate the photochemopreventive effect of topical formulations containing rutin SLNs, against ultraviolet B (UVB) radiation, inducing sunburn cells, caspase-3, cyclobutane pyrimidine dimers, lipid peroxidation, and metalloproteinase formation. The rutin SLNs presented average size of 74.22 ± 2.77 nm, polydispersion index of 0.16 ± 0.04, encapsulation efficiency of 98.90 ± 0.25%, and zeta potential of -53.0 ± 1.61 mV. The rutin SLNs were able to efficiently protect against UVB induced in the analysed parameters in both skin models. Furthermore, the rutin SLNs inhibited lipid peroxidation and metalloproteinase formation. These results support the use of rutin SLNs as skin photochemopreventive agents for topical application to the skin.

Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells.

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, ß-catenin, GSK-3ß and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.

Faecal carriage of multidrug-resistant bacteria and associated risk factors: results from a point prevalence study.

OBJECTIVES: Since 2003, incidences of carbapenemase-producing Gram-negative bacilli (CP-GNB) and vancomycin-resistant Enterococcus faecium (VRE) have steadily increased in France. We therefore conducted a point prevalence study to estimate carriage rates of CP-GNB, VRE and ESBL-producing Enterobacterales (ESBL-PE) and associated risk factors. METHODS: Between September 2019 and January 2020, all inpatients hospitalized on a given day in 11 teaching hospitals in the Paris urban area were eligible. Patient interviews and rectal swab screening results were recorded by dedicated nurses. The swabs were plated onto selective chromogenic media and processed using the GeneXpert® system. RESULTS: Of 2396 patients, 364 (15.2%) yielded at least one multiresistant bacterial isolate, including 29 CP-GNB carriers (1.2%), 13 VRE carriers (0.5%) and 338 ESBL-PE carriers (14%). In 15 patients (4.4% of ESBL-PE carriers and 36.6% of CP-GNB/VRE carriers), concomitant CP-GNB/VRE and ESBL-PE carriage was observed. In 7/29 CP-GNB and 7/13 VRE carriers, carbapenemase production and vanA in the screening samples was only detected with Xpert® tests. The OXA-48 gene was predominant in 13/34 CP-GNB isolates from 29 carriers. From the 338 ESBL-PE carriers, 372 isolates were recovered, mainly Escherichia coli (61.2%). Among 379 children, 1.1% carried a CP-GNB/VRE strain, and 12.4% carried an ESBL strain. Previous hospitalization outside mainland France, previous antimicrobial treatment and previous ESBL-PE carriage were the main risk factors associated with CP-GNB and/or VRE carriage. CONCLUSIONS: The low CP-GNB and VRE prevalence likely reflects the French policy to limit intrahospital spread of CP-GNB and VRE strains.

Release of infectious virus and cytokines in nasopharyngeal swabs from individuals infected with non-alpha or alpha SARS-CoV-2 variants: an observational retrospective study.

BACKGROUND: The dynamics of SARS-CoV-2 alpha variant shedding and immune responses at the nasal mucosa remain poorly characterised. METHODS: We measured infectious viral release, antibodies and cytokines in 426 PCR+ nasopharyngeal swabs from individuals harboring non-alpha or alpha variants. FINDINGS: With both lineages, viral titers were variable, ranging from 0 to >106 infectious units. Rapid antigenic diagnostic tests were positive in 94% of samples with infectious virus. 68 % of individuals carried infectious virus within two days after onset of symptoms. This proportion decreased overtime. Viable virus was detected up to 14 days. Samples containing anti-spike IgG or IgA did not generally harbor infectious virus. Ct values were slightly but not significantly lower with alpha. This variant was characterized by a fast decrease of infectivity overtime and a marked release of 13 cytokines (including IFN-b, IP-10 and IL-10). INTERPRETATION: The alpha variant displays modified viral decay and cytokine profiles at the nasopharyngeal mucosae during symptomatic infection. FUNDING: This retrospective study has been funded by Institut Pasteur, ANRS, Vaccine Research Institute, Labex IBEID, ANR/FRM and IDISCOVR, Fondation pour la Recherche Médicale.

Surviving an Extensive Burn Injury Using Advanced Skin Replacement Technologies.

There have been significant improvements in the technology available for treating extensive burns in the past decade. This case presents two unique, skin replacement technologies that were used to treat an 86% surface area flame burn in a pediatric patient. A temporary dermal replacement, known as "Novosorb™ Biodegradable Temporizing Matrix" was first used to stabilize the burn injury and remained in place for approximately 3 months. Given the large burn size and lack of available donor skin for grafting, a permanent skin replacement product known as "Self-Assembled Skin Substitute (SASS)" was then utilized to cover the burns. SASS is a novel technology that was developed to replace skin as an autologous skin graft and is currently available in Canada through a clinical trial for major burns. Ultimately, the concurrent use of these two technologies allowed for the unprecedented survival of a child following an extensive and life-threatening burn injury.

Three-Dimensional Model of Hypertrophic Scar Using a Tissue-Engineering Approach.

Following wound healing, skin is replaced by a specialized tissue called scar. Sometime, this scar can become pathologic, called hypertrophic scar, with a high amount of extracellular matrix, capillaries, and myofibroblast persistence. To understand the mechanisms at the origin of the fibrosis is paramount to treat patients, but despite few animal models and in vitro studies using mainly human pathological cells cultured on plastic on monolayer, the treatment of these fibrotic scars remains unsatisfactory. As in tissue, cells are most often imbedded in extracellular matrix, we have developed, using a tissue engineering method, new in vitro models to study human fibrotic skin pathologies as hypertrophic scars. Human cells isolated from hypertrophic scars are used to reconstitute a three-dimensional fibrotic skin comprising both dermal and epidermal parts. This method called the self-assembly approach is based on the cell capacity to reconstitute their own environment as in vivo. In this chapter, the described methods include extraction and culture of human scar keratinocytes and fibroblasts from cutaneous biopsies as well as the protocols to produce fibrotic skin that can be used to study pathological process.

Granulation tissue myofibroblasts during normal and pathological skin healing: The interaction between their secretome and the microenvironment.

The first role that was proposed for the myofibroblasts located in skin granulation tissue was to contract the edges of the wound in order to reduce the surface to be repaired. This role, linked to the presence of alpha smooth muscle actin, was very quickly confirmed and is part of the definition of granulation tissue myofibroblasts. However, myofibroblasts are cells that also play a much more central role in wound healing. Indeed, it has been shown that these cells produce large quantities of matrix components, and that they stimulate angiogenesis and can recruit immune cells. These actions take place via the secretion of molecules into their environment or indirectly via the production of microvesicles containing pro-fibrotic and pro-angiogenic molecules. Pathologically, granulation tissue can develop into a hypertrophic scar that histologically looks like granulation tissue, but which can remain for months or even years. It has been hypothesized that the myofibroblasts in these tissues remained present instead of disappearing by apoptosis, causing the maintenance of granulation tissue rather than allowing its change into a mature scar. Understanding the roles of both pathological and healthy myofibroblasts in wound tissue is crucial in order to better intervene in the healing mechanism.

GPR43 regulates sodium butyrate-induced angiogenesis and matrix remodeling.

Butyrate is a short-chain fatty acid (SCFA) derived from microbiota and is involved in a range of cell processes in a concentration-dependent manner. Low concentrations of sodium butyrate (NaBu) were shown to be proangiogenic. However, the mechanisms associated with these effects are not yet fully known. Here, we investigated the contribution of the SCFA receptor GPR43 in the proangiogenic effects of local treatment with NaBu and its effects on matrix remodeling using the sponge-induced fibrovascular tissue model in mice lacking the Gpr43 gene (Gpr43-KO) and the wild-type (WT) mice. We demonstrated that NaBu (0.2 mM intraimplant) treatment enhanced the neovascularization process, blood flow, and VEGF levels in a GPR43-dependent manner in the implants. Moreover, NaBu was able to modulate matrix remodeling aspects of the granulation tissue such as proteoglycan production, collagen deposition, and α-smooth muscle actin (α-SMA) expression in vivo, besides increasing transforming growth factor (TGF)-ß1 levels in the fibrovascular tissue, in a GPR43-dependent manner. Interestingly, NaBu directly stimulated L929 murine fibroblast migration and TGF-ß1 and collagen production in vitro. GPR43 was found to be expressed in human dermal fibroblasts, myofibroblasts, and endothelial cells. Overall, our findings evidence that the metabolite-sensing receptor GPR43 contributes to the effects of low dose of NaBu in inducing angiogenesis and matrix remodeling during granulation tissue formation. These data provide important insights for the proposition of new therapeutic approaches based on NaBu, beyond the highly explored intestinal, anti-inflammatory, and anticancer purposes, as a local treatment to improve tissue repair, particularly, by modulating granulation tissue components.NEW & NOTEWORTHY Our data show the contribution of the metabolite-sensing receptor GPR43 in the effects of low dose of sodium butyrate (NaBu) on stimulating angiogenesis and extracellular matrix remodeling in a model of granulation tissue formation in mice. We also show that human dermal fibroblasts, myofibroblasts, and endothelial cells express the receptor GPR43. These data provide important insights for the use of NaBu in local therapeutic approaches applicable to tissue repair in sites other than the intestine.

Shedding of proangiogenic microvesicles from hypertrophic scar myofibroblasts.

Hypertrophic scars are a common complication of burn injuries and represent a major challenge in terms of prevention and treatment. These scars are characterized by a supraphysiological vascular density and by the presence of pathological myofibroblasts (Hmyos) displaying a low apoptosis propensity. However, the nature of the association between these two hallmarks of hypertrophic scarring remains largely unexplored. Here, we show that Hmyos produce signalling entities known as microvesicles that significantly increase the three cellular processes underlying blood vessel formation: endothelial cell proliferation, migration and assembly into capillary-like structures. The release of microvesicles from Hmyos was dose-dependently induced by the serum protein α-2-macroglobulin. Using flow cytometry, we revealed the presence of the α-2-macroglobulin receptor-low-density lipoprotein receptor-related protein 1-on the surface of Hmyos. The inhibition of the binding of α-2-macroglobulin to its receptor abolished the shedding of proangiogenic microvesicles from Hmyos. These findings suggest that the production of microvesicles by Hmyos contributes to the excessive vascularization of hypertrophic scars. α-2-Macroglobulin modulates the release of these microvesicles through interaction with low-density lipoprotein receptor-related protein 1.

Biofabrication and preclinical evaluation of a large-sized human self-assembled skin substitute.

Severe skin burns are widely treated using split-thickness skin autografts. However, the accessibility of the donor site may be limited depending on the size of the injured surface. As an alternative to skin autografts, our laboratory is clinically investigating a model of human self-assembled skin substitute (SASS) with a standard size of 35 cm2. For the management of extensive skin wounds, multiple grafts are required to cover the entire wound bed. Even if SASSs could provide an adequate and efficient treatment, in some cases, the long-term follow-up of the skin graft site reveals the appearance of marks at the junction between SASSs. This study aims to produce a large-sized self-assembled skin substitute (L-SASS; 289 cm2) and evaluate its preclinical potential for skin wound coverage. The L-SASSs and SASSs shared similar contraction behavior on an agar surface, thickness, and epidermal differentiation in vitro. After grafting, similar histological results were obtained for skin substitutes produced with both methods. Hence, the self-assembly approach of tissue engineering is a scaffold-free method that allows the production of living skin substitutes in a large format.

Novel Foot-and-Mouth Disease Vaccine Platform: Formulations for Safe and DIVA-Compatible FMD Vaccines With Improved Potency.

Inactivated, wild-type foot-and-mouth disease virus (FMDV) vaccines are currently used to control FMD around the world. These traditional FMD vaccines are produced using large quantities of infectious, virulent, wild-type FMD viruses, with the associated risk of virus escape from manufacturing facilities or incomplete inactivation during the vaccine formulation process. While higher quality vaccines produced from wild-type FMDV are processed to reduce non-structural antigens, there is still a risk that small amounts of non-structural proteins may be present in the final product. A novel, antigenically marked FMD-LL3B3D vaccine platform under development by Zoetis, Inc. and the USDA-ARS, consists of a highly attenuated virus platform containing negative antigenic markers in the conserved non-structural proteins 3Dpol and 3B that render resultant vaccines fully DIVA compatible. This vaccine platform allows for the easy exchange of capsid coding sequences to create serotype-specific vaccines. Here we demonstrate the efficacy of the inactivated FMD-LL3B3D-A24 Cruzeiro vaccine in cattle against wild-type challenge with A24 Cruzerio. A proprietary adjuvant system was used to formulate the vaccines that conferred effective protection at low doses while maintaining the DIVA compatibility. In contrast to wild-type FMDV, the recombinant FMD-LL3B3D mutant viruses have been shown to induce no clinical signs of FMD and no shedding of virus in cattle or pigs when inoculated as a live virus. The FMD-LL3B3D vaccine platform, currently undergoing development in the US, provides opportunities for safer vaccine production with full DIVA compatibility in support of global FMDV control and eradication initiatives.

PLGF-1 contained in normal wound myofibroblast-derived microvesicles stimulated collagen production by dermal fibroblasts.

During the last stages of wound healing, myofibroblasts differentiate mainly from fibroblasts. Myofibroblasts from normal skin wounds (Wmyo) can communicate with its surrounding using secreted factors. They also have the capacity to produce microvesicles (MVs), a type of extracellular vesicles, as mediators of intercellular communication. MVs cargo are potentially capable of regulating the behavior of targeted cells and tissues. The aim of this study is to evaluate the effect of Wmyo-derived MVs on dermal fibroblasts and to determine the responsible signaling molecule. Microvesicles were obtained from culture media of myofibroblasts and characterized using protein quantification, dynamic light scattering and transmission electron microscopy. Uptake of fluorescent MVs in fibroblasts was assessed by flow cytometry. Cytokines concentrations were quantified in MV samples by a multiplex ELISA. Different concentration of MVs or a selected cytokine were used as treatments over fibroblasts culture for 5 days. Following the treatments, parameters linked to the extracellular matrix were studied. Lastly, the selected cytokine was neutralized within MVs before evaluating collagen production. We showed that Wmyo derived-MVs were internalized by dermal fibroblasts. Cytokine array analysis revealed that a large amount of placental growth factor 1 (PLGF-1) (0.88 ± 0.63 pg/µg proteins in MVs) could be detected in MVs samples. Cutaneous fibroblasts treated with MVs or PLGF-1 showed significantly stimulated procollagen I level production (Fold change of 1.80 ± 0.18 and 2.07 ± 0.18, respectively). Finally, the neutralization of PLGF-1 in MVs significantly inhibited the production of procollagen I by fibroblasts. Our study shows that Wmyo derived-MVs are involved in intercellular communication by stimulating collagen production by fibroblasts during wound healing. This effect is possibly attained through PLGF-1 signalling. These findings represent a promising opportunity to gain insight into how MVs and Wmyo may mediate the healing of a skin wound.