Neurotoxic reactive astrocytes induce cell death via saturated lipids.

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease1-6. Here we report an approach to isolate one component of the long-sought astrocyte-derived toxic factor5,6. Notably, instead of a protein, saturated lipids contained in APOE and APOJ lipoparticles mediate astrocyte-induced toxicity. Eliminating the formation of long-chain saturated lipids by astrocyte-specific knockout of the saturated lipid synthesis enzyme ELOVL1 mitigates astrocyte-mediated toxicity in vitro as well as in a model of acute axonal injury in vivo. These results suggest a mechanism by which astrocytes kill cells in the central nervous system.

Proteomic profiling of interferon-responsive reactive astrocytes in rodent and human.

Astrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called "reactivity." Reactive astrocytes exhibit distinct and context-dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub-state defined by interferon-responsive genes like Igtp, Ifit3, Mx1, and others, called interferon-responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub-state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC-differentiated astrocytes using TNF, IL1α, C1Q, and IFNß and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.

One Scaffold, Different Organelle Sensors: pH-Activable Fluorescent Probes for Targeting Live Microglial Cell Organelles.

Targeting live cell organelles is essential for imaging, understanding, and controlling specific biochemical processes. Typically, fluorescent probes with distinct structural scaffolds are used to target specific cell organelles. Here, we have designed a modular one-step synthetic strategy using a common reaction intermediate to develop new lysosomal, mitochondrial, and nucleus-targeting pH-activable fluorescent probes that are all based on a single boron dipyrromethane scaffold. The divergent cell organelle targeting was achieved by synthesizing probes with specific functional group changes to the central scaffold resulting in differential fluorescence and pKa . Specifically, we show that the functional group transformation of the same scaffold influences cellular localization and specificity of pH-activable fluorescent probes in live primary microglial cells with pKa values ranging from ∼3.2-6.0. We introduce a structure-organelle-relationship (SOR) framework to target nuclei (NucShine), lysosomes (LysoShine), and mitochondria (MitoShine) in live microglia. This work will result in future applications of SOR beyond imaging to target and control organelle-specific biochemical processes in disease-specific models.

Prevalence and phenotype associations of complement factor I mutations in geographic atrophy.

Rare variants in the complement factor I (CFI) gene, associated with low serum factor I (FI) levels, are strong risk factors for developing the advanced stages of age-related macular degeneration (AMD). No studies have been undertaken on the prevalence of disease-causing CFI mutations in patients with geographic atrophy (GA) secondary to AMD. A multicenter, cross-sectional, noninterventional study was undertaken to identify the prevalence of pathogenic rare CFI gene variants in an unselected cohort of patients with GA and low FI levels. A genotype-phenotype study was performed. Four hundred and sixty-eight patients with GA secondary to AMD were recruited to the study, and 19.4% (n = 91) demonstrated a low serum FI concentration (below 15.6 µg/ml). CFI gene sequencing on these patients resulted in the detection of rare CFI variants in 4.7% (n = 22) of recruited patients. The prevalence of CFI variants in patients with low serum FI levels and GA was 25%. Of the total patients recruited, 3.2% (n = 15) expressed a CFI variant classified as pathogenic or likely pathogenic. The presence of reticular pseudodrusen was detected in all patients with pathogenic CFI gene variants. Patients with pathogenic CFI gene variants and low serum FI levels might be suitable for FI supplementation in therapeutic trials.

Management of Destructive Colon Injuries after Damage Control Surgery.

After the World War II, fecal diversion became the standard of care for colon injuries, although medical, logistic, and technical advancements have challenged this approach. Damage control surgery serves to temporize immediately life-threatening conditions, and definitive management of destructive colon injuries is delayed until after appropriate resuscitation. The bowel can be left in discontinuity for up to 3 days before edema ensues, but the optimal repair window remains within 12 to 48 hours. Delayed anastomosis performed at the take-back operation or stoma formation has been reported with variable results. Studies have revealed good outcomes in those undergoing anastomosis after damage control surgery; however, they point to a subgroup of trauma patients considered to be "high risk" that may benefit from fecal diversion. Risk factors influencing morbidity and mortality rates include hypotension, massive transfusion, the degree of intra-abdominal contamination, associated organ injuries, shock, left-sided colon injury, and multiple comorbid conditions. Patients who are not suitable for anastomosis by 36 hours after damage control may be best managed with a diverting stoma. Failures are more likely related to ongoing instability, and the management strategy of colorectal injury should be based mainly on the patient's overall condition.

Neutrophil derived microparticles increase mortality and the counter-inflammatory response in a murine model of sepsis.

Although advances in medical care have significantly improved sepsis survival, sepsis remains the leading cause of death in the ICU. This is likely due to a lack of complete understanding of the pathophysiologic mechanisms that lead to dysfunctional immunity. Neutrophil derived microparticles (NDMPs) have been shown to be the predominant microparticle present at infectious and inflamed foci in human models, however their effect on the immune response to inflammation and infection is sepsis has not been fully elucidated. As NDMPs may be a potential diagnostic and therapeutic target, we sought to determine the impact NDMPs on the immune response to a murine polymicrobial sepsis. We found that peritoneal neutrophil numbers, bacterial loads, and NDMPs were increased in our abdominal sepsis model. When NDMPs were injected into septic mice, we observed increased bacterial load, decreased neutrophil recruitment, increased expression of IL-10 and worsened mortality. Furthermore, the NDMPs express phosphatidylserine and are ingested by F4/80 macrophages via a Tim-4 and MFG-E8 dependent mechanism. Finally, upon treatment, NDMPs decrease macrophage activation, increase IL-10 release and decrease macrophage numbers. Altogether, these data suggest that NDMPs enhance immune dysfunction in sepsis by blunting the function of neutrophils and macrophages, two key cell populations involved in the early immune response to infection. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Impact of caspase-8 and PKA in regulating neutrophil-derived microparticle generation.

The morbidity and mortality from sepsis continues to remain high despite extensive research into understanding this complex immunologic process. Further, while source control and antibiotic therapy have improved patient outcomes, many immunologically based therapies have fallen short. Microparticles (MPs) are intact vesicles that serve as mediators of intercellular communication as well as markers of inflammation in various disease processes. We have previously demonstrated that MPs can be produced at the infected foci during sepsis, are predominantly of neutrophil derivation (NDMPs) and can modulate immune cells. In this study, we sought to elucidate the molecular mechanisms underlying NDMP generation. Using thioglycolate (TGA) to recruit and activate neutrophils, we first determined that intra-peritoneal TGA increase NDMP accumulation. We next utilized TGA-elicited neutrophils in vitro to investigate signaling intermediates involved in NDMP production, including the intrinsic and extrinsic caspase pathways, cAMP dependent PKA and Epac activation as well as the role myosin light chain kinase (MLCK) as a final mediator of NDMP release. We observed that NDMP generation was dependent on the extrinsic caspase apoptotic pathway (caspase 3 and caspase 8), cAMP activation of PKA but not of Epac, and on activation of MLCK. Altogether, these data contribute to an overall framework depicting the molecular mechanisms that regulate NDMP generation.

Association Between Opioid Abuse/Dependence and Outcomes in Hospitalized Heart Failure Patients.

Opioid use is associated with unintentional and intentional overdose and is one of the leading causes of emergency room visits and accidental deaths. However, the association between opioid abuse/dependence and outcomes in hospitalized patients has not been well studied. Congestive heart failure (HF) is the fourth most common cause of hospitalization in the United States. The purpose of this study was to examine the effect of opioid abuse/dependence on outcomes in patients hospitalized with HF. We queried the 2002-2010 Nationwide Inpatient Sample databases to identify all patients aged 18 years and older admitted with the primary diagnosis of HF. Multivariate logistic regression analysis was used to compare the frequency of hospital-acquired conditions (HACs) and in-hospital mortality between patients with and without a history of opioid abuse/dependence. Of 9,993,240 patients with HF, 29,014 had a history of opioid abuse or dependence. Opioid abusers/dependents were likely to be younger men of poor socioeconomic background with self pay or Medicaid as their primary payer. They had a lower prevalence of dyslipidemia, diabetes mellitus, coronary artery disease, prior myocardial infarction, and peripheral vascular disease (P < 0.001 for all). They were more likely to be smokers and have chronic pulmonary disease, depression, liver disease, and obesity (P < 0.001 for all). Patients with a history of opioid abuse/dependence had lower incidence of HACs (14.8% vs. 16.5%, adjusted odds ratio: 0.71, P < 0.001) and lower in-hospital mortality (1.3% vs. 3.6%, adjusted odds ratio: 0.64, P < 0.001) as compared with patients without prior opioid abuse/dependence. In conclusion, among adult patients aged 18 years and older hospitalized with HF, opioid abuse/dependence was associated with lower frequency of HACs and lower in-hospital mortality.

Thymic stromal lymphopoietin mediates the host response and increases mortality during sepsis.

BACKGROUND: Sepsis and subsequent multiorgan system failure is associated with high rates of mortality and morbidity. Thymic stromal lymphopoietin (TSLP) is a cytokine that can be produced by keratinocytes and epithelial cells. Primarily, TSLP has been shown to promote counter-inflammatory processes. However, its potential expression or role in the pathogenesis of sepsis is largely unexplored. We hypothesized that TSLP is expressed during sepsis and TSLP blockade would alter the immune response and mortality. MATERIALS AND METHODS: Mice underwent cecal ligation and puncture (CLP) to produce a physiologically relevant murine model for sepsis. Cohorts were either treated with neutralizing TSLP antibodies or isotype controls before the CLP to determine changes in survival, bacterial loads, cytokine levels, and neutrophil function. RESULTS: It was observed that TSLP levels peaked at 6 h and remained detectable up to 48 h after CLP. Mice pretreated with neutralizing TSLP showed decreased mortality and bacterial load after CLP. Additionally, we determined that septic mice pretreated with the anti-TSLP antibody had increased tumor necrosis factor alpha and oxidative burst as well as increased interleukin 17 and neutrophil numbers compared with mice pretreated with isotype controls. CONCLUSIONS: TSLP levels peak early but are sustained during the first 48 h of sepsis. We speculate that TSLP blunts the neutrophil response resulting in increased bacterial load and mortality.

Trauma surgical skill sustainment at the University of Chicago AMEDD Military-Civilian Trauma Team Training Site: an observation report.

Background: The Army Medical Department (AMEDD) Military-Civilian Trauma Team Training (AMCT3) Program was developed to enhance the trauma competency and capability of the medical force by embedding providers at busy civilian trauma centers. Few reports have been published on the outcomes of this program since its implementation. Methods: The medical and billing records for the two AMCT3 embedded trauma surgeons at the single medical center were retrospectively reviewed for care provided during August 2021 through July 2022. Abstracted data included tasks met under the Army's Individual Critical Task List (ICTL) for general surgeons. The Knowledge, Skills, and Abilities (KSA) score was estimated based on previously reported point values for procedures. To assess for successful integration of the embedded surgeons, data were also abstracted for two newly hired civilian trauma surgeons. Results: The annual clinical activity for the first AMCT3 surgeon included 444 trauma evaluations and 185 operative cases. The operative cases included 80 laparotomies, 15 thoracotomies, and 15 vascular exposures. The operative volume resulted in a KSA score of 21 998 points. The annual clinical activity for the second AMCT3 surgeon included 424 trauma evaluations and 194 operative cases. The operative cases included 92 laparotomies, 8 thoracotomies, and 25 vascular exposures. The operative volume resulted in a KSA score of 22 799 points. The first civilian surgeon's annual clinical activity included 453 trauma evaluations and 151 operative cases, resulting in a KSA score of 16 738 points. The second civilian surgeon's annual clinical activity included 206 trauma evaluations and 96 operative cases, resulting in a KSA score of 11 156 points. Conclusion: The AMCT3 partnership at this single center greatly exceeds the minimum deployment readiness metrics established in the ICTLs and KSAs for deploying general surgeons. The AMEDD experience provided a deployment-relevant case mix with an emphasis on complex vascular injury repairs.

Mechanisms underlying mouse TNF-α stimulated neutrophil derived microparticle generation.

Despite advances in understanding and treatment of sepsis, it remains a disease with high mortality. Neutrophil Derived Microparticles (NDMPs) are present during sepsis and can modulate the immune system. As TNF-α is a cytokine that predominates in the initial stages of sepsis, we evaluated whether and how TNF-α can induce NDMPs in mice. We observed that TNF-α treatment results in increased NDMP numbers. We also determined that the activation of either TNF receptor 1 (TNFr1) or TNF receptor 2 (TNFr2) resulted in increased NDMP numbers and that activation of both resulted in an additive increase. Inhibition of Caspase 8 diminishes NDMPs generated through TNFr1 activation and inhibition of NF-κB abrogates NDMPs generated through activation of both TNFr1 and TNFr2. We conclude that the early production of TNF-α during sepsis can increase NDMP numbers through activation of the Caspase 8 pathway or NF-κB.

Interleukin-7 ameliorates immune dysfunction and improves survival in a 2-hit model of fungal sepsis.

BACKGROUND: Secondary hospital-acquired fungal infections are common in critically-ill patients and mortality remains high despite antimicrobial therapy. Interleukin-7 (IL-7) is a potent immunotherapeutic agent that improves host immunity and has shown efficacy in bacterial and viral models of infection. This study examined the ability of IL-7, which is currently in multiple clinical trials (including hepatitis and human immunodeficiency virus), to improve survival in a clinically relevant 2-hit model of fungal sepsis. METHODS: Mice underwent cecal ligation and puncture to induce peritonitis. Four days later, surviving mice had intravenous injection with Candida albicans. Following Candida infection, mice were treated with IL-7 or saline control. The effect of IL-7 on host immunity and survival was recorded. RESULTS: IL-7 ameliorated the loss of immune effector cells and increased lymphocyte functions, including activation, proliferation, expression of adhesion molecules, and interferon-γ production. These beneficial effects of IL-7 were associated with an increase in global immunity as reflected by an enhanced delayed type hypersensitivity response and a 1.7-fold improvement in survival. CONCLUSIONS: The present findings showing that IL-7 improves survival in fungal sepsis, together with its previously reported efficacy in bacterial and viral infectious models, further supports its use as a novel immunotherapeutic in sepsis.

Transarterial interventions in civilian gunshot wound injury: experience from a level-1 trauma center.

PURPOSE: To assess the effectiveness of trans-arterial vascular interventions in treatment of civilian gunshot wounds (GSW). MATERIALS AND METHODS: A retrospective review was performed at a level-1 trauma center to include 46 consecutive adults admitted due to GSW related hemorrhage and treated with endovascular interventions from July 2018 to July 2022. Patient demographics and procedural metrics were retrieved. Primary outcomes of interest include technical success and in-hospital mortality. Factors of mortality were assessed using a logistic regression model. RESULTS: Twenty-one patients were brought to the endovascular suite directly (endovascular group) from the trauma bay and 25 patients after treatment in the operating room (OR group). The OR group had higher hemodynamic instability (48.0% vs 19.0%, p = 0.040), lower hemoglobin (12.9 vs 10.1, p = 0.001) and platelet counts (235.2 vs 155.1, p = 0.003), and worse Acute Physiology and Chronic Health Evaluation (APACHE) score (4.1 vs 10.2, p < 0.0001) at the time of initial presentation. Technical success was achieved in all 40 cases in which targeted embolization was attempted (100%). Empiric embolization was performed in 6/46 (13.0%) patients based on computed tomographic angiogram (CTA) and operative findings. Stent-grafts were placed in 3 patients for subclavian artery injuries. Availability of pre-intervention CTA was associated with shorter fluoroscopy time (19.8 ± 12.1 vs 30.7 ± 18.6 min, p = 0.030). A total of 41 patients were discharged in stable condition (89.1%). Hollow organ injury was associated with mortality (p = 0.039). CONCLUSION: Endovascular embolization and stenting were effective in managing hemorrhage due to GSW in a carefully selected population. Hollow organ injury was a statistically significant predictor of mortality. Pre-intervention CTA enabled targeted, shorter and equally effective procedures.

Amyloid ß Induces Lipid Droplet-Mediated Microglial Dysfunction in Alzheimer's Disease.

Several microglia-expressed genes have emerged as top risk variants for Alzheimer's disease (AD). Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD-risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. Here we show that microglia form lipid droplets (LDs) upon exposure to amyloid-beta (Aß), and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. LD formation is dependent upon age and disease progression and is more prominent in the hippocampus in mice and humans. Despite variability in LD load between microglia from male versus female animals and between cells from different brain regions, LD-laden microglia exhibited a deficit in Aß phagocytosis. Unbiased lipidomic analysis identified a substantial decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TAGs) as the key metabolic transition underlying LD formation. We demonstrate that DGAT2, a key enzyme for the conversion of FFAs to TAGs, promotes microglial LD formation, is increased in microglia from 5xFAD and human AD brains, and that inhibiting DGAT2 improved microglial uptake of Aß. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

Proteomic Alterations and Novel Markers of Neurotoxic Reactive Astrocytes in Human Induced Pluripotent Stem Cell Models.

Astrocytes respond to injury, infection, and inflammation in the central nervous system by acquiring reactive states in which they may become dysfunctional and contribute to disease pathology. A sub-state of reactive astrocytes induced by proinflammatory factors TNF, IL-1α, and C1q ("TIC") has been implicated in many neurodegenerative diseases as a source of neurotoxicity. Here, we used an established human induced pluripotent stem cell (hiPSC) model to investigate the surface marker profile and proteome of TIC-induced reactive astrocytes. We propose VCAM1, BST2, ICOSL, HLA-E, PD-L1, and PDPN as putative, novel markers of this reactive sub-state. We found that several of these markers colocalize with GFAP+ cells in post-mortem samples from people with Alzheimer's disease. Moreover, our whole-cells proteomic analysis of TIC-induced reactive astrocytes identified proteins and related pathways primarily linked to potential engagement with peripheral immune cells. Taken together, our findings will serve as new tools to purify reactive astrocyte subtypes and to further explore their involvement in immune responses associated with injury and disease.

The Effect of Inorganic and Organic Zinc Supplementation on Growth Performance, Mineral Profile and Gene Expression Pattern of GLUT1 in Malabari Kids.

The objective of this experiment was to study and compare the effects of dietary supplementation of organic and inorganic zinc (Zn) on growth performance, nutrient utilisation and gene expression pattern of glucose transporter protein in peripheral blood mononuclear cells (PBMC) in Malabari kids. Fifteen, 3-4-month-old goat kids were divided into three groups uniformly by using completely randomised design (CRD). Group G1 was fed on basal diet as per NRC requirement, and G2 and G3 were fed on basal diet + 40 ppm Zn as inorganic zinc sulphate (ZnSO4) and 40 ppm Zn as organic Zn methionine, respectively, for a period of 91 days. Supplementation of inorganic and organic Zn had no significant effect on dry matter (DM) intake. The digestibility of crude protein (CP), ether extract (EE), neutral detergent fibre (NDF), hemicellulose and cellulose was significantly more in the organic Zn-supplemented group. The average daily gain and feed:gain ratio were significantly (p < 0.05) better in group G3 in comparison to G1 and G2, while the nitrogen retention was found to be (p < 0.01) higher in group G3 than in group G1. Zinc balance was found to be significantly (p < 0.01) increased in both supplemented groups with respect to unsupplemented group G1. The blood glucose level was (p < 0.01) lower in group G3 compared to group G1 suggesting the insulin-like activity of Zn. Serum Zn concentration was significantly (p < 0.01) increased in both Zn-supplemented groups. There was a significant (p < 0.05) rise in glucose transporter GLUT1 expression in groups G2 and G3 when compared to control group G1. Moreover, GLUT1 expression was found to be higher (p < 0.05) in group G3 as against the animals of group G2. Lowered blood glucose level might have stimulated more glucose transporter GLUT1 expression in PBMC. Organic Zn supplemented at 40 ppm level resulted in better growth performance, nutrient digestibility and nitrogen as well as Zn retention in goat kids. There was better absorption, and hence, less amount of Zn got excreted in the organic Zn-supplemented group.

The Ethics and Politics of Gun Violence Research.

Gun violence is an epidemic that affects hundreds of thousands of Americans each year. Despite gun violence being disproportionately more lethal than other leading causes of trauma, there is a dearth of research being carried out on its root causes and prevention strategies. For the past 20 years, lobbying and politics have interfered with the forward progress of gun violence research. Physicians have a history of producing actionable public-health change and have an ethical obligation to fight for the research that will benefit their patients.

Monitoring phagocytic uptake of amyloid ß into glial cell lysosomes in real time.

Phagocytosis by glial cells is essential to regulate brain function during health and disease. Therapies for Alzheimer's disease (AD) have primarily focused on targeting antibodies to amyloid ß (Aß) or inhibitng enzymes that make it, and while removal of Aß by phagocytosis is protective early in AD it remains poorly understood. Impaired phagocytic function of glial cells during later stages of AD likely contributes to worsened disease outcome, but the underlying mechanisms of how this occurs remain unknown. We have developed a human Aß1-42 analogue (AßpH) that exhibits green fluorescence upon internalization into the acidic organelles of cells but is non-fluorescent at physiological pH. This allowed us to image, for the first time, glial uptake of AßpH in real time in live animals. We find that microglia phagocytose more AßpH than astrocytes in culture, in brain slices and in vivo. AßpH can be used to investigate the phagocytic mechanisms responsible for removing Aß from the extracellular space, and thus could become a useful tool to study Aß clearance at different stages of AD.