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Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to the activation of microglia. Accurate identification of microglial populations is key to understanding therapeutic approaches that modify microglial responses to TBI and improve long-term outcome measures. Notably, previous studies often utilized an outdated convention to describe microglial phenotypes. We conducted a temporal analysis of the response to controlled cortical impact (CCI) in rat microglia between ipsilateral and contralateral hemispheres across seven time points, identified microglia through expression of activation markers including CD45, CD11b/c, and p2y12 receptor and evaluated their activation state using additional markers of CD32, CD86, RT1B, CD200R, and CD163. We identified unique sub-populations of microglial cells that express individual or combination of activation markers across time points. We further portrayed how the size of these sub-populations changes through time, corresponding to stages in TBI response. We described longitudinal changes in microglial population after CCI in two different locations using activation markers, showing clear separation into cellular sub-populations that feature different temporal patterns of markers after injury. These changes may aid in understanding the symptomatic progression following TBI and help define microglial subpopulations beyond the outdated M1/M2 paradigm.

For several decades, multipotent mesenchymal stromal cells (MSCs) have been extensively studied for their therapeutic potential across a wide range of diseases. In the preclinical setting, MSCs demonstrate consistent ability to promote tissue healing, down-regulate excessive inflammation and improve outcomes in animal models. Several proposed mechanisms of action have been posited and demonstrated across an array of models. However, translation into clinical practice has proven considerably more difficult. A number of prominent well-funded late-phase clinical trials have failed, thus calling out for new efforts to optimize product delivery in the clinical setting. In this review, we discuss novel topics critical to the successful translation of MSCs from pre-clinical to clinical applications. In particular, we focus on the major routes of cell delivery, aspects related to hemocompatibility, and potential safety concerns associated with MSC therapy in the different settings.

Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus’ assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives. Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Here, the authors elucidate the precise time-regulated secretion of mussel adhesive proteins in Perna viridis , probing their surface structures and subsequent roles.

Biomineralization, the process by which mineralized tissues grow and harden via biogenic mineral deposition, is a relatively lengthy process in many mineral-producing organisms, resulting in challenges to study the growth and biomineralization of complex hard mineralized tissues. Arthropods are ideal model organisms to study biomineralization because they regularly molt their exoskeletons and grow new ones in a relatively fast timescale, providing opportunities to track mineralization of entire tissues. Here, we monitored the biomineralization of the mantis shrimp dactyl club—a model bioapatite-based mineralized structure with exceptional mechanical properties—immediately after ecdysis until the formation of the fully functional club and unveil an unusual development mechanism. A flexible membrane initially folded within the club cavity expands to form the new club’s envelope. Mineralization proceeds inwards by mineral deposition from this membrane, which contains proteins regulating mineralization. Building a transcriptome of the club tissue and probing it with proteomic data, we identified and sequenced Club Mineralization Protein 1 (CMP-1), an abundant mildly phosphorylated protein from the flexible membrane suggested to be involved in calcium phosphate mineralization of the club, as indicated by in vitro studies using recombinant CMP-1. This work provides a comprehensive picture of the development of a complex hard tissue, from the secretion of its organic macromolecular template to the formation of the fully functional club.

The only available option to treat radiation-induced hematopoietic syndrome is allogeneic hematopoietic cell transplantation, a therapy unavailable to many patients undergoing treatment for malignancy, which would also be infeasible in a radiological disaster. Stromal cells serve as critical components of the hematopoietic stem cell niche and are thought to protect hematopoietic cells under stress. Prior studies that have transplanted mesenchymal stromal cells (MSCs) without co-administration of a hematopoietic graft have shown underwhelming rescue of endogenous hematopoiesis and have delivered the cells within 24 h of radiation exposure. Herein, we examine the efficacy of a human bone marrow-derived MSC therapy delivered at 3 h or 30 h in ameliorating radiation-induced hematopoietic syndrome and show that pancytopenia persists despite MSC therapy. Animals exposed to radiation had poorer survival and experienced loss of leukocytes, platelets, and red blood cells. Importantly, mice that received a therapeutic dose of MSCs were significantly less likely to die but experienced equivalent collapse of the hematopoietic system. The cause of the improved survival was unclear, as complete blood counts, splenic and marrow cellularity, numbers and function of hematopoietic stem and progenitor cells, and frequency of niche cells were not significantly improved by MSC therapy. Moreover, human MSCs were not detected in the bone marrow. MSC therapy reduced crypt dropout in the small intestine and promoted elevated expression of growth factors with established roles in gut development and regeneration, including PDGF-A, IGFBP-3, IGFBP-2, and IGF-1. We conclude that MSC therapy improves survival not through overt hematopoietic rescue but by positive impact on other radiosensitive tissues, such as the intestinal mucosa. Collectively, these data reveal that MSCs could be an effective countermeasure in cancer patients and victims of nuclear accidents but that MSCs alone do not significantly accelerate or contribute to recovery of the blood system.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Traumatic brain injury (TBI) results in activated microglia. Activated microglia can be measured in vivo by using positron emission topography (PET) ligand peripheral benzodiazepine receptor standardized uptake values (PBR28suv). Cell based therapies have utilized autologous bone marrow mononuclear cells (BMMNCs) to attenuate activated microglia after TBI. This study aims to utilize in vivo PBR28suv to assess the efficacy of BMMNCs therapy after TBI. Seventy-two hours after CCI injury, BMMNCs were harvested from the tibia and injected via tail-vein at 74 h after injury at a concentration of 2 million cells per kilogram of body weight. There were three groups of rats: Sham, CCI-alone and CCI-BMMNCs (AUTO). One hundred twenty days after injury, rodents were imaged with PBR28 and their cognitive behavior assessed utilizing the Morris Water Maze. Subsequent ex vivo analysis included brain volume and immunohistochemistry. BMMNCs therapy attenuated PBR28suv in comparison to CCI alone and it improved spatial learning as measured by the Morris Water Maze. Ex vivo analysis demonstrated preservation of brain volume, a decrease in amoeboid-shaped microglia in the dentate gyrus and an increase in the ratio of ramified to amoeboid microglia in the thalamus. PBR28suv is a viable option to measure efficacy of BMMNCs therapy after TBI.

Dengue is a widespread viral disease with 3.6 billion people at risk worldwide. Humanized monoclonal antibody (mAb) 513, currently undergoing clinical trials in Singapore, targets an epitope on the envelope protein domain III exposed at the surface of the viral particle. This antibody potently neutralizes all four dengue virus serotypes in a humanized mouse model that recapitulates human dengue infection, without signs of antibody-mediated enhancement of the disease. The crystal structure of single-chain variable fragment (scFv) 513 bound to the envelope protein domain III from dengue virus serotype 4 was used as a template to explore the molecular origins of the broader cross-reactivity and increased in vivo potency of mAb 513, compared to the parent murine mAb 4E11, using molecular dynamics simulations and network analyses. These two methods are a powerful complement to existing structural and binding data and detail specific interactions that underpin the differential binding of the two antibodies. We found that a Glu at position H55 (Glu H55 ) from the second Complementarity Determining Region of the Heavy chain (CDR-H2) which corresponds to Ala in 4E11, is a major contributor to the enhancement in the interactions of mAb 513 compared to 4E11. Importantly, we also validate the importance of Glu H55 using site-directed mutagenesis followed by isothermal titration calorimetry measurements.

Traumatic brain injury (TBI) causes a profound inflammatory response within the central nervous system and peripheral immune system, which contributes to secondary brain injury and further morbidity and mortality. Preclinical investigations have demonstrated that treatments that downregulate microglia activation and polarize them toward a reparative/anti‐inflammatory phenotype have improved outcomes in preclinical models. However, no therapy to date has translated into proven benefits in human patients. Regulatory T cells (Treg) have been shown to downregulate pathologic immune responses of the innate and adaptive immune system across a variety of pathologies. Furthermore, cellular therapy has been shown to augment host Treg responses in preclinical models; yet, studies investigating the use of Treg as a therapeutic for TBI are lacking. In a rodent TBI model, we demonstrate that human umbilical cord blood Treg modulate the central and peripheral immune response after injury in vitro and in vivo. A rat model of traumatic brain injury was used to determine whether regulatory T cells (Treg) could be used to treat neuroinflammation associated with severe central nervous system trauma. We utilized multicolor flow cytometry 30d after injury to rapidly assess the changes in the phenotype of microglia, which were visualized using a dimensional reduction algorithm (t‐distributed stochastic neighbor embedding). This method clearly maps populations that change as a result of injury but respond to Treg treatment. Further analysis demonstrates that these particular populations express microglia markers that change in expression levels in response to injury, including CD11bc (pictured here). This response is ameliorated by Treg treatment 24 hours after injury.

The immune system plays critical roles in promoting tissue repair during recovery from neurotrauma but is also responsible for unchecked inflammation that causes neuronal cell death, systemic stress, and lethal immunodepression. Understanding the immune response to neurotrauma is an urgent priority, yet current models of traumatic brain injury (TBI) inadequately recapitulate the human immune response. Here, we report the first description of a humanized model of TBI and show that TBI places significant stress on the bone marrow. Hematopoietic cells of the marrow are regionally decimated, with evidence pointing to exacerbation of underlying graft-versus-host disease (GVHD) linked to presence of human T cells in the marrow. Despite complexities of the humanized mouse, marrow aplasia caused by TBI could be alleviated by cell therapy with human bone marrow mesenchymal stromal cells (MSCs). We conclude that MSCs could be used to ameliorate syndromes triggered by hypercytokinemia in settings of secondary inflammatory stimulus that upset marrow homeostasis such as TBI. More broadly, this study highlights the importance of understanding how underlying immune disorders including immunodepression, autoimmunity, and GVHD might be intensified by injury.