Injury causes a systemic inflammatory response syndrome (SIRS) clinically much like sepsis 1. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors 2. Similarly, cellular injury can release endogenous damage-associated molecular patterns (DAMPs) that activate innate immunity 3. Mitochondria are evolutionary endosymbionts that were derived from bacteria 4 and so might bear bacterial molecular motifs. We show here that injury releases mitochondrial DAMPs (MTD) into the circulation with functionally important immune consequences. MTD include formyl peptides and mitochondrial DNA. These activate human neutrophils (PMN) through formyl peptide receptor-1 and TLR9 respectively. MTD promote PMN Ca2+ flux and phosphorylation of MAP kinases, thus leading to PMN migration and degranulation in vitro and in vivo. Circulating MTD can elicit neutrophil-mediated organ injury. Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These can then signal through identical innate immune pathways to create a sepsis-like state. The release of such mitochondrial ‘enemies within’ by cellular injury is a key link between trauma, inflammation and SIRS.

Trauma and sepsis can cause acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) in part by triggering neutrophil (PMN)-mediated increases in endothelial cell (EC) permeability. We had shown that mitochondrial (mt) damage-associated molecular patterns (DAMPs) appear in the blood after injury or shock and activate human PMN. So we now hypothesized that mitochondrial DAMPs (MTD) like mitochondrial DNA (mtDNA) and peptides might play a role in increased EC permeability during systemic inflammation and proceeded to evaluate the underlying mechanisms. MtDNA induced changes in EC permeability occurred in two phases: a brief, PMN-independent ‘spike’ in permeability was followed by a prolonged PMN-dependent increase in permeability. Fragmented mitochondria (MTD) caused PMN-independent increase in EC permeability that were abolished with protease treatment. Exposure to mtDNA caused PMN-EC adherence by activating expression of adherence molecule expression in both cell types. Cellular activation was manifested as an increase in PMN calcium flux and EC MAPK phosphorylation. Permeability and PMN adherence were attenuated by endosomal TLR inhibitors. EC lacked formyl peptide receptors but were nonetheless activated by mt-proteins, showing that non-formylated mt-protein DAMPs can activate EC. Mitochondrial DAMPs can be released into the circulation by many processes that cause cell injury and lead to pathologic endothelial permeability. We show here that mitochondria contain multiple DAMP motifs that can act on EC and/or PMN via multiple pathways. This can enhance PMN adherence to EC, activate PMN-EC interactions and subsequently increase systemic endothelial permeability. Mitochondrial DAMPs may be important therapeutic targets in conditions where inflammation pathologically increases endothelial permeability.

Hypothesis-Fractures and femoral reaming are associated with lung injury. The mechanisms linking fractures and inflammation are unclear; but tissue disruption might release mitochondria. Mitochondria are evolutionarily derived from bacteria and contain "Damage Associated Molecular Patterns" (DAMPs) like formylated peptides that can activate immunocytes. We therefore studied whether fracture reaming releases mitochondrial DAMPs (MTD) and how MTD act on immune cells.Methods-Femur fracture reamings (FFx) from 10 patients were spun to remove bone particulates. Supernatants were assayed for mitochondrial DNA (mtDNA). Mitochondria were isolated from the residual reaming slurry, sonicated and spun at 12,000g. The resultant MTD were assayed for their ability to cause neutrophil (PMN) Ca 2+ transient production, p44/42 MAPK phosphorylation, IL-8 release and matrix metalloproteinase-9 (MMP9) release with and without formyl peptide receptor-1 (FPR1) blockade. Rats were injected with MTD and whole lung assayed for p44/42 activation.Results-mtDNA appears at many thousand fold normal plasma levels in FFx and at intermediate levels in patients' plasma, suggesting release from fracture to plasma. FFx MTD caused brisk PMN Ca 2+ flux, activated PMN p44/42 MAPK and caused PMN release of IL-8 and MMP9. Responses to MTD were inhibited by FPR1 blockade using Cyclosporin H and anti-FPR1. MTD injection caused P44/42 phosphorylation in rat lung.Conclusions-FFx reaming releases mitochondria into the wound and circulation. MTD then activates PMN. Release of damage signals like MTD from FFx may underlie activation of the cytokine cascades known to be associated with facture fixation and lung injury. KeywordsInnate immunity; formyl peptides; fractures; neutrophils Acute lung injury and adult respiratory distress syndrome (ALI/ARDS) occur after fractures in a sporadic entity often termed "fat embolism syndrome" (FES). FES is hard to distinguish from ALI/ARDS occurring after sepsis, and may be associated with reamed nailing more than other methods of fixation. Current concepts emphasize that fracture hematomas are rich in inflammatory mediators 1 -4 that can activate immune cells like neutrophils (PMN) that can injure the lung but it is unknown what the primary events are causing fractures to be rich in mediators. Understanding the events linking mechanical injury to immune organ dysfunction is essential if effective therapies are to be developed. MATERIALS AND METHODS Research complianceStudies were performed under the supervision of the Institutional Review Board (IRB) of Beth Israel Deaconess Medical Center (BIDMC) and Harvard Medical School. Fracture reaming specimens were collected under waiver of consent for discarded materials. Consent was obtained for sampling and archiving of trauma plasma samples from the patients or their legally authorized representative whenever such consent was available. Animal experimentation was approved by the IACUC of BIDMC. Patients and biologic samplesFemoral reamings were collected intra-operati...

Systemic inflammatory response syndrome (SIRS) is a fundamental host response common to bacterial infection and sterile tissue injury. SIRS can cause organ dysfunction and death but its mechanisms are incompletely understood. Moreover, SIRS can progress to organ failure or death despite being sterile or after control of the inciting infection. Biomarkers discriminating between sepsis, sterile SIRS and post-infective SIRS would therefore help direct care. Circulating mitochondrial DNA (mtDNA) is a damage-associated molecular pattern (DAMP) reflecting cellular injury. Circulating bacterial 16S-DNA (bDNA) is a pathogen-associated pattern (PAMP) reflecting ongoing infection. We developed qPCR assays to quantify these markers and predicted their plasma levels might help distinguish sterile injury from infection. To study these events in primates we assayed banked serum from papio baboons that had undergone a brief challenge of intravenous Bacillus anthracis deltaSterne (modified to remove toxins) followed by antibiotics (anthrax) that causes organ failure and death. To investigate the progression of sepsis to “severe” sepsis and death we studied animals where anthrax was pretreated with drotrecogin alfa (aPC), which attenuates sepsis in baboons. We also contrasted lethal anthrax bacteremia against non-lethal E.coli bacteremia and against sterile tissue injury from Shiga-like toxin-1 (Stx1). bDNA and mtDNA levels in timed samples were correlated with blood culture results and assays of organ function. Sterile injury by Stx1 increased mtDNA but bDNA was undetectable: consistent with the absence of infection. The bacterial challenges caused parallel early bDNA and mtDNA increases, but bDNA detected pathogens even after bacteria were undetectable by culture. Sub-lethal E.coli challenge only caused transient rises in mtDNA consistent with a self-limited injury. In lethal anthrax challenge (n=4) bDNA increased transiently but mtDNA levels remained elevated until death, consistent with persistent septic tissue damage after bacterial clearance. Critically, aPC pre-treatment (n=4) allowed mtDNA levels to decay after bacterial clearance with sparing of organ function and survival. In summary, host tissue injury correlates with mtDNA whether infective or sterile. mtDNA and bDNA PCRs can quantify tissue injury incurred by septic or sterile mechanisms and suggest the source of SIRS of unknown origin.

BACKGROUND AND PURPOSE: Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infection is associated with hypercoagulability. We sought to evaluate the demographic and clinical characteristics of cerebral venous thrombosis among patients hospitalized for coronavirus disease 2019 (COVID-19) at 6 tertiary care centers in the New York City metropolitan area. MATERIALS AND METHODS:We conducted a retrospective multicenter cohort study of 13,500 consecutive patients with COVID-19 who were hospitalized between March 1 and May 30, 2020. RESULTS: Of 13,500 patients with COVID-19, twelve had imaging-proved cerebral venous thrombosis with an incidence of 8.8 per 10,000 during 3 months, which is considerably higher than the reported incidence of cerebral venous thrombosis in the general population of 5 per million annually. There was a male preponderance (8 men, 4 women) and an average age of 49 years (95% CI, 36-62 years; range, 17-95 years). Only 1 patient (8%) had a history of thromboembolic disease. Neurologic symptoms secondary to cerebral venous thrombosis occurred within 24 hours of the onset of the respiratory and constitutional symptoms in 58% of cases, and 75% had venous infarction, hemorrhage, or both on brain imaging. Management consisted of anticoagulation, endovascular thrombectomy, and surgical hematoma evacuation. The mortality rate was 25%. CONCLUSIONS:Early evidence suggests a higher-than-expected frequency of cerebral venous thrombosis among patients hospitalized for COVID-19. Cerebral venous thrombosis should be included in the differential diagnosis of neurologic syndromes associated with SARS-CoV-2 infection. ABBREVIATIONS: COVID-19 ¼ coronavirus disease 2019; CVST ¼ cerebral venous sinus thrombosis; CVT ¼ cerebral venous thrombosis; SARS-CoV-2 ¼ Severe Acute Respiratory Syndrome coronavirus 2 C oronavirus disease 2019 (COVID-19) is predominantly an acute respiratory disease caused by a single-stranded RNA virus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, China. 1 The virus possesses a spike protein that binds to angiotensin-converting enzyme receptors, expressed on respiratory epithelium, facilitating entry into the host cell. [2][3][4] Susceptibility of organ systems to this virus may depend on the extent of expression of angiotensin-converting enzyme receptors on cell surfaces. These receptors are expressed on endothelial cells, pericytes, macrophages, glial cells, and cardiac myocytes. [2][3][4] Viral entry into these cells can lead to diverse manifestations such as acute respiratory distress syndrome, acute kidney injury, transaminitis, cardiac injury, and neurologic complications. [3][4][5][6] Neurologic symptoms include headache, confusion, hypogeusia, hyposmia, myalgias, and delirium, while neurologic complications include acute ischemic stroke, encephalitis, and Guillain-Barre syndrome. 3,[6][7][8] Postmortem data have revealed cerebral edema and partial neuronal degeneration in some patients as well. 9 Early evidence suggests an inc...

Bacterial DNA (bDNA) contains hypo-methylated “CpG” repeats that can be recognized by toll-like receptor (TLR)-9 as a pathogen-associated molecular pattern (PAMP). The ability of bDNA to initiate lung injury via TLR-9 has been inferred on the basis of studies using artificial CpG DNA. But the role of authentic bDNA in lung injury is still unknown. Moreover, the mechanisms by which CpG DNA species can lead to pulmonary injury are unknown, although neutrophils (PMN) are thought to play a key role in the genesis of septic acute lung injury (ALI). We evaluated the effects of bDNA on PMN-endothelial cell (EC) interactions thought critical for initiation of ALI. Using a bio-capacitance system to monitor real-time changes in endothelial permeability, we demonstrate here that bDNA causes EC permeability in a dose-dependent manner uniquely in the presence of PMN. These permeability changes are inhibited by chloroquine, suggesting TLR9-dependency. When PMN were pre-incubated with bDNA and applied to EC or when bDNA was applied to EC without PMN, no permeability changes were detected. To study the underlying mechanisms we evaluated the effects of bDNA on PMN-EC adherence. bDNA significantly increased PMN adherence to EC in association with up-regulated adhesion molecules in both cell types. Taken together, our results strongly support the conclusion that bDNA can initiate lung injury by stimulating PMN-EC adhesive interactions predisposing to endothelial permeability. bDNA stimulation of TLR9 appears to promote enhanced gene expression of adhesion molecules in both cell types. This leads to PMN-EC cross-talk which is required for injury to occur.

Background and Purpose: Acute ischemic stroke (AIS) is a rare occurrence during pregnancy and the postpartum period. Existing literature evaluating endovascular mechanical thrombectomy (MT) for this patient population is limited. Methods: The National Inpatient Sample was queried from 2012 to 2018 to identify and characterize pregnant and postpartum patients (up to 6 weeks following childbirth) with AIS treated with MT. Complications and outcomes were compared with nonpregnant female patients treated with MT and to other pregnant and postpartum patients managed medically. Complex samples regression models and propensity score matching were implemented to assess adjusted associations and to address confounding by indication, respectively. Results: Among 4590 pregnant and postpartum patients with AIS, 180 (3.9%) were treated with MT, and rates of utilization increased following the MT clinical trial era (2015–2018; 1.9% versus 5.3%, P =0.011). Compared with nonpregnant patients with AIS treated with MT, they experienced lower rates of intracranial hemorrhage (11% versus 24%, P =0.069) and poor functional outcome (50% versus 72%, P =0.003) at discharge. Pregnant/postpartum status was independently associated with a lower likelihood of development of intracranial hemorrhage (adjusted odds ratio, 0.26 [95% CI, 0.09–0.70]; P =0.008) following multivariable analysis adjusting for age, illness severity, and stroke severity. Following propensity score matching, pregnant and postpartum patients treated with MT and those medically managed differed in frequency of venous thromboembolism (17% versus 0%, P =0.001) and complications related to pregnancy (44% versus 64%, P =0.034), but not in functional outcome at discharge or hospital length of stay. Pregnant and postpartum women treated with MT did not experience mortality or miscarriage during hospitalization. Conclusions: This large-scale analysis utilizing national claims data suggests that MT is a safe and efficacious therapy for AIS during pregnancy and the postpartum period. In the absence of prospective clinical trials, population-based cross-sectional analyses such as the present study provide valuable clinical insight.

Glioblastoma (GB) is a highly aggressive and infiltrative brain tumor characterized by poor outcomes and a high rate of recurrence despite maximal safe resection, chemotherapy, and radiation. Superparamagnetic iron oxide nanoparticles (SPIONs) are a novel tool that can be used for many applications including magnetic targeting, drug delivery, gene delivery, hyperthermia treatment, cell tracking, or multiple simultaneous functions. SPIONs are studied as a magnetic resonance imaging tumor contrast agent by targeting tumor cell proteins or tumor vasculature. Drug delivery to GB tumor has been targeted with SPIONs in murine models. In addition to targeting tumor cells for imaging or drug-delivery, SPION has also been shown to be effective at targeting for hyperthermia. Along with animal models, human trials have been conducted for a number of different modes of SPION utilization, with important findings and lessons for further preclinical and clinical experiments. SPIONs are opening up several new avenues for monitoring and treatment of GB tumors; here, we review the current research and a variety of possible clinical applications.Glioblastoma (GB) represents the most common and most aggressive type of primary brain tumor in adults with a nearly uniform fatal outcome. Despite multimodal therapy, including maximal safe surgical resection of the tumor with adjuvant chemotherapy and radiation, the overall survival time remains about 15 months (1). GB tumors are heterogeneous with genetic and epigenetic variation within the tumor mass. These aspects make the development of therapies to eradicate the entire tumor a challenging task. Conventional therapeutic strategies include surgery followed by chemotherapy and radiation. Some chemotherapy regimens may include temozolomide, monoclonal antibodies against vascular endothelial growth factor (VEGF), or inhibitors of tyrosine kinase receptors (1, 2). Poor outcome and recurrence is largely attributed to: a) the highly aggressive and infiltrative nature of GB tumors which may increase the likelihood of subtotal resection; b) limited delivery of therapeutics across the bloodbrain barrier (BBB); and c) glioma stem cells that contribute to formation, expansion, recurrence, and therapy resistance resulting in tumor recurrence (3).To enhance the efficacy of treatment and drug delivery, several strategies have been developed. One strategy focuses on the utilization of superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs in brain cancer treatment are an attractive modality for several reasons. They can be used for magnetic targeting, drug delivery, gene delivery, hyperthermia treatment, and cell tracking (4-9).The SPION core is usually made of magnetite (Fe 3 O 4 ) or maghemite (γ-Fe 2 O 3 ), and the surface of superparamagnetic core is covered with a compatible coating such as dextran, polyethylene glycol (PEG), poly-L-Lysine, D-mannose, or other different polymers to prevent agglomeration and to enhance biocompatibility (10-13). Active molecules can be bound to the coating, w...