Mesenchymal Stromal Cells Facilitate Neutrophil-Trained Immunity by Reprogramming Hematopoietic Stem Cells.

Novel therapeutics are urgently needed to prevent opportunistic infections in immunocompromised individuals undergoing cancer treatments or other immune-suppressive therapies. Trained immunity is a promising strategy to reduce this burden of disease. We previously demonstrated that mesenchymal stromal cells (MSCs) preconditioned with a class A CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) agonist, can augment emergency granulopoiesis in a murine model of neutropenic sepsis. Here, we used a chimeric mouse model to demonstrate that MSCs secrete paracrine factors that act on lineage-negative c-kit+ hematopoietic stem cells (HSCs), leaving them "poised" to enhance emergency granulopoiesis months after transplantation. Chimeric mice developed from HSCs exposed to conditioned media from MSCs and CpG-ODN-preconditioned MSCs showed significantly higher bacterial clearance and increased neutrophil granulopoiesis following lung infection than control mice. By Cleavage Under Targets and Release Using Nuclease (CUT&RUN) chromatin sequencing, we identified that MSC-conditioned media leaves H3K4me3 histone marks in HSCs at genes involved in myelopoiesis and in signaling persistence by the mTOR pathway. Both soluble factors and extracellular vesicles from MSCs mediated these effects on HSCs and proteomic analysis by mass spectrometry revealed soluble calreticulin as a potential mediator. In summary, this study demonstrates that trained immunity can be mediated by paracrine factors from MSCs to induce neutrophil-trained immunity by reprogramming HSCs for long-lasting functional changes in neutrophil-mediated antimicrobial immunity.

Animal model of repeated low-level blast traumatic brain injury displays acute and chronic neurobehavioral and neuropathological changes.

Blast-induced neurotrauma (BINT) is not only a signature injury to soldiers in combat field and training facilities but may also a growing concern in civilian population due to recent increases in the use of improvised explosives by insurgent groups. Unlike moderate or severe BINT, repeated low-level blast (rLLB) is different in its etiology as well as pathology. Due to the constant use of heavy weaponry as part of combat readiness, rLLB usually occurs in service members undergoing training as part of combat readiness. rLLB does not display overt pathological symptoms; however, earlier studies report chronic neurocognitive changes such as altered mood, irritability, and aggressive behavior, all of which may be caused by subtle neuropathological manifestations. Current animal models of rLLB for investigation of neurobehavioral and neuropathological alterations have not been adequate and do not sufficiently represent rLLB conditions. Here, we developed a rat model of rLLB by applying controlled low-level blast pressures (<10 psi) repeated successively five times to mimic the pressures experienced by service members. Using this model, we assessed anxiety-like symptoms, motor coordination, and short-term memory as a function of time. We also examined levels of superoxide-producing enzyme NADPH oxidase, microglial activation, and reactive astrocytosis as factors likely contributing to these neurobehavioral changes. Animals exposed to rLLB displayed acute and chronic anxiety-like symptoms, motor and short-term memory impairments. These changes were paralleled by increased microglial activation and reactive astrocytosis. Conversely, animals exposed to a single low-level blast did not display significant changes. Collectively, this study demonstrates that, unlike a single low-level blast, rLLB exerts a cumulative impact on different brain regions and produces chronic neuropathological changes in so doing, may be responsible for neurobehavioral alterations.

Central and peripheral auditory abnormalities in chinchilla animal model of blast-injury.

Exposure to blast overpressure or high-intensity sound can cause injuries to the auditory system, which leads to hearing loss or tinnitus. In this study, we examined the involvement of peripheral auditory system (PAS), and central auditory system (CAS) changes after exposure to blast overpressure (15-25 psi) on Day 1 and additionally during 7 days of post blast time period in chinchillas. Auditory brainstem response (ABR), distortion product otoacoustic emission (DPOAE), and cochlear hair cell changes were measured or identified in post-blast period within 7 days to detect injuries in the PAS. In the CAS, changes in NMDAR1 (excitatory receptor) and GABAA (inhibitory receptor) as well as changes in serotonin (5-HT2A) and acetylcholine (AChR) receptors were examined in different brain regions: auditory cortex (AC), geniculate body (GB), inferior colliculus (IC) and amygdala by immunofluorescence staining. We observed the PAS abnormalities of increased ABR threshold and decreased DPOAE response in animals after blast exposure with hearing protection devices (e.g., earplug). Blast exposure also caused a reduction in both NMDAR1 and GABAA receptor levels in acute condition (post-blast or Day 1) in AC and IC, while serotonin and acetylcholine receptor levels displayed a biphasic response at Day 1 and Day 7 post-exposure. Results demonstrate that the earplug can protect the tympanic membrane and middle ear against structural damage, but the hearing level, cochlear outer hair cell, and the central auditory system (levels of excitatory and inhibitory neurotransmitter receptors) were only partially protected at the tested blast overpressure level. The findings in this study indicate that blast exposure can cause both peripheral and central auditory dysfunctions, and the central auditory response is independent of peripheral auditory damage. The CAS dysfunction is likely mediated by direct transmission of shockwaves in all the regions of central nervous system (CNS), including nerves and surrounding tissues along the auditory pathways. Hence, targeting central auditory neurotransmitter abnormalities may have a therapeutic benefit to attenuate blast-induced hearing loss and tinnitus.

Synergistic Role of Oxidative Stress and Blood-Brain Barrier Permeability as Injury Mechanisms in the Acute Pathophysiology of Blast-induced Neurotrauma.

Blast-induced traumatic brain injury (bTBI) has been recognized as the common mode of neurotrauma amongst military and civilian personnel due to an increased insurgent activity domestically and abroad. Previous studies from our laboratory have identified enhanced blood-brain barrier (BBB) permeability as a significant, sub-acute (four hours post-blast) pathological change in bTBI. We also found that NADPH oxidase (NOX)-mediated oxidative stress occurs at the same time post-blast when the BBB permeability changes. We therefore hypothesized that oxidative stress is a major causative factor in the BBB breakdown in the sub-acute stages. This work therefore examined the role of NOX1 and its downstream effects on BBB permeability in the frontal cortex (a region previously shown to be the most vulnerable) immediately and four hours post-blast exposure. Rats were injured by primary blast waves in a compressed gas-driven shock tube at 180 kPa and the BBB integrity was assessed by extravasation of Evans blue and changes in tight junction proteins (TJPs) as well as translocation of macromolecules from blood to brain and vice versa. NOX1 abundance was also assessed in neurovascular endothelial cells. Blast injury resulted in increased extravasation and reduced levels of TJPs in tissues consistent with our previous observations. NOX1 levels were significantly increased in endothelial cells followed by increased superoxide production within 4 hours of blast. Blast injury also increased the levels/activation of matrix metalloproteinase 3 and 9. To test the role of oxidative stress, rats were administered apocynin, which is known to inhibit the assembly of NOX subunits and arrests its function. We found apocynin completely inhibited dye extravasation as well as restored TJP levels to that of controls and reduced matrix metalloproteinase activation in the sub-acute stages following blast. Together these data strongly suggest that NOX-mediated oxidative stress contributes to enhanced BBB permeability in bTBI through a pathway involving increased matrix metalloproteinase activation.

Microglia Receptors in Animal Models of Traumatic Brain Injury.

Microglia have been implicated as a key mediator of chronic inflammation following traumatic brain injury (TBI). The animal models of TBI vary significantly based on the type of brain injury (focal versus diffuse). This has made it extremely difficult to assess the role of microglia and the window of microglia activation. Hence, the focus of this review is to summarize the time course of microglia activation in various animal models of TBI. The review explores the repertoire of secondary injury mechanisms such as aberrant neurotransmitter release, oxidative stress, blood-brain barrier disruption, and production of pro-inflammatory cytokines that follow microglia activation. Since receptors act as sensors for activation, we highlight certain microglia receptors that have been implicated in TBI pathology, including fractalkine receptor (CX3CR1), purinergic receptor (P2Y12R), Toll-like receptor (TLR4), scavenger receptors, tumor necrosis factor receptor (TNF-1R), interleukin receptor (IL-1R), complement receptors, and peroxisome proliferator-activated receptor (PPAR). In addition to describing their downstream signaling pathways in TBI, we describe the functional consequences of their activation and the implication in behavioral outcomes. Taken together, this review will provide a holistic view of the role of microglia and its receptors in TBI based on animal studies.

Temporal and Spatial Effects of Blast Overpressure on Blood-Brain Barrier Permeability in Traumatic Brain Injury.

Blast-induced traumatic brain injury (bTBI) is a "signature wound" in soldiers during training and in combat and has also become a major cause of morbidity in civilians due to increased insurgency. This work examines the role of blood-brain barrier (BBB) disruption as a result of both primary biomechanical and secondary biochemical injury mechanisms in bTBI. Extravasation of sodium fluorescein (NaF) and Evans blue (EB) tracers were used to demonstrate that compromise of the BBB occurs immediately following shock loading, increases in intensity up to 4 hours and returns back to normal in 24 hours. This BBB compromise occurs in multiple regions of the brain in the anterior-posterior direction of the shock wave, with maximum extravasation seen in the frontal cortex. Compromise of the BBB is confirmed by (a) extravasation of tracers into the brain, (b) quantification of tight-junction proteins (TJPs) in the brain and the blood, and (c) tracking specific blood-borne molecules into the brain and brain-specific proteins into the blood. Taken together, this work demonstrates that the BBB compromise occurs as a part of initial biomechanical loading and is a function of increasing blast overpressures.

A Single Primary Blast-Induced Traumatic Brain Injury in a Rodent Model Causes Cell-Type Dependent Increase in Nicotinamide Adenine Dinucleotide Phosphate Oxidase Isoforms in Vulnerable Brain Regions.

Blast-induced traumatic brain injury (bTBI) is a leading cause of morbidity in soldiers on the battlefield and in training sites with long-term neurological and psychological pathologies. Previous studies from our laboratory demonstrated activation of oxidative stress pathways after blast injury, but their distribution among different brain regions and their impact on the pathogenesis of bTBI have not been explored. The present study examined the protein expression of two isoforms: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and 2 (NOX1, NOX2), corresponding superoxide production, a downstream event of NOX activation, and the extent of lipid peroxidation adducts of 4-hydroxynonenal (4HNE) to a range of proteins. Brain injury was evaluated 4 h after the shock-wave exposure, and immunofluorescence signal quantification was performed in different brain regions. Expression of NOX isoforms displayed a differential increase in various brain regions: in hippocampus and thalamus, there was the highest increase of NOX1, whereas in the frontal cortex, there was the highest increase of NOX2 expression. Cell-specific analysis of changes in NOX expression with respect to corresponding controls revealed that blast resulted in a higher increase of NOX1 and NOX 2 levels in neurons compared with astrocytes and microglia. Blast exposure also resulted in increased superoxide levels in different brain regions, and such changes were reflected in 4HNE protein adduct formation. Collectively, this study demonstrates that primary blast TBI induces upregulation of NADPH oxidase isoforms in different regions of the brain parenchyma and that neurons appear to be at higher risk for oxidative damage compared with other neural cells.

Computing with liquid crystal fingers: models of geometric and logical computation.

When a voltage is applied across a thin layer of cholesteric liquid crystal, fingers of cholesteric alignment can form and propagate in the layer. In computer simulation, based on experimental laboratory results, we demonstrate that these cholesteric fingers can solve selected problems of computational geometry, logic, and arithmetics. We show that branching fingers approximate a planar Voronoi diagram, and nonbranching fingers produce a convex subdivision of concave polygons. We also provide a detailed blueprint and simulation of a one-bit half-adder functioning on the principles of collision-based computing, where the implementation is via collision of liquid crystal fingers with obstacles and other fingers.