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Background Despite the high morbidity and mortality associated with sepsis, the relationship between the plasma proteome and clinical outcome is poorly understood. In this study, we used targeted plasma proteomics to identify novel biomarkers of sepsis in critically ill patients. Methods Blood was obtained from 15 critically ill patients with suspected/confirmed sepsis (Sepsis-3.0 criteria) on intensive care unit (ICU) Day-1 and Day-3, as well as age- and sex-matched 15 healthy control subjects. A total of 1161 plasma proteins were measured with proximal extension assays. Promising sepsis biomarkers were narrowed with machine learning and then correlated with relevant clinical and laboratory variables. Results The median age for critically ill sepsis patients was 56 (IQR 51–61) years. The median MODS and SOFA values were 7 (IQR 5.0–8.0) and 7 (IQR 5.0–9.0) on ICU Day-1, and 4 (IQR 3.5–7.0) and 6 (IQR 3.5–7.0) on ICU Day-3, respectively. Targeted proteomics, together with feature selection, identified the leading proteins that distinguished sepsis patients from healthy control subjects with ≥ 90% classification accuracy; 25 proteins on ICU Day-1 and 26 proteins on ICU Day-3 (6 proteins overlapped both ICU days; PRTN3, UPAR, GDF8, NTRK3, WFDC2 and CXCL13). Only 7 of the leading proteins changed significantly between ICU Day-1 and Day-3 (IL10, CCL23, TGFα1, ST2, VSIG4, CNTN5, and ITGAV; P Conclusions Targeted proteomics with feature selection identified proteins altered in critically ill sepsis patients relative to healthy control subjects. Correlations between protein expression and clinical/laboratory variables were identified, each providing pathophysiological insight. Our exploratory data provide a rationale for further hypothesis-driven sepsis research.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is a global health care emergency. Anti-SARS-CoV-2 serological profiling of critically ill COVID-19 patients was performed to determine their humoral response. Blood was collected from critically ill ICU patients, either COVID-19 positive (+) or COVID-19 negative (−), to measure anti-SARS-CoV-2 immunoglobulins: IgM, IgA, IgG, and Total Ig (combined IgM/IgA/IgG). Cohorts were similar, with the exception that COVID-19+ patients had a greater body mass indexes, developed bilateral pneumonias more frequently and suffered increased hypoxia when compared to COVID-19- patients (p &lt, 0.05). The mortality rate for COVID-19+ patients was 50%. COVID-19 status could be determined by anti-SARS-CoV-2 serological responses with excellent classification accuracies on ICU day 1 (89%), ICU day 3 (96%), and ICU days 7 and 10 (100%). The importance of each Ig isotype for determining COVID-19 status on combined ICU days 1 and 3 was: Total Ig, 43%, IgM, 27%, IgA, 24% and IgG, 6%. Peak serological responses for each Ig isotype occurred on different ICU days (IgM day 13 &gt, IgA day 17 &gt, IgG persistently increased), with the Total Ig peaking at approximately ICU day 18. Those COVID-19+ patients who died had earlier or similar peaks in IgA and Total Ig in their ICU stay when compared to patients who survived (p &lt, 0.005). Critically ill COVID-19 patients exhibit anti-SARS-CoV-2 serological responses, including those COVID-19 patients who ultimately died, suggesting that blunted serological responses did not contribute to mortality. Serological profiling of critically ill COVID-19 patients may aid disease surveillance, patient cohorting and help guide antibody therapies such as convalescent plasma.

Background COVID19 is caused by the SARS-CoV-2 virus and has been associated with severe inflammation leading to organ dysfunction and mortality. Our aim was to profile the transcriptome in leukocytes from critically ill patients positive for COVID19 compared to those negative for COVID19 to better understand the COVID19-associated host response. For these studies, all patients admitted to our tertiary care intensive care unit (ICU) suspected of being infected with SARS-CoV-2, using standardized hospital screening methodologies, had blood samples collected at the time of admission to the ICU. Transcriptome profiling of leukocytes via ribonucleic acid sequencing (RNAseq) was then performed and differentially expressed genes as well as significantly enriched gene sets were identified. Results We enrolled seven COVID19 + (PCR positive, 2 SARS-CoV-2 genes) and seven age- and sex-matched COVID19- (PCR negative) control ICU patients. Cohorts were well-balanced with the exception that COVID19− patients had significantly higher total white blood cell counts and circulating neutrophils and COVID19 + patients were more likely to suffer bilateral pneumonia. The mortality rate for this cohort of COVID19 + ICU patients was 29%. As indicated by both single-gene based and gene set (GSEA) approaches, the major disease-specific transcriptional responses of leukocytes in critically ill COVID19 + ICU patients were: (i) a robust overrepresentation of interferon-related gene expression; (ii) a marked decrease in the transcriptional level of genes contributing to general protein synthesis and bioenergy metabolism; and (iii) the dysregulated expression of genes associated with coagulation, platelet function, complement activation, and tumour necrosis factor/interleukin 6 signalling. Conclusions Our findings demonstrate that critically ill COVID19 + patients on day 1 of admission to the ICU display a unique leukocyte transcriptional profile that distinguishes them from COVID19− patients, providing guidance for future targeted studies exploring novel prognostic and therapeutic aspects of COVID19.

Background COVID-19 is associated with a novel multi-system inflammatory syndrome that shares some characteristics with Kawasaki’s Disease. The syndrome manifestation is delayed relative to COVID-19 onset, with a spectrum of clinical severity. Clinical signs may include persistent fever, gastrointestinal symptoms, cardiac inflammation and/or shock. Case Presentation: We measured 59 inflammatory and endothelial injury plasma analytes in an adolescent girl that presented with malaise, fever, cough, strawberry tongue and jaundice. Her COVID-19 status was positive with detection of 2 SARS-CoV-2 viral genes using polymerase chain reaction. She was treated with intravenous immunoglobulin prior to blood draw, but our plasma measurements suggested a unique analyte expression pattern associated with inflammation, endothelial injury and microvascular glycocalyx degradation. Conclusions COVID-19 is associated with a multi-system inflammatory syndrome and a unique inflammatory and endothelial injury signature. Analyte markers of inflammation and endothelium injury might serve as putative biomarkers and/or be investigated further as potential therapeutic targets.

Background: COVID19 is caused by SARS-CoV-2 infection to which there is no community immunity Patients admitted to intensive care units (ICU) have high mortali

OBJECTIVE: Diabetic ketoacidosis in children is associated with vasogenic cerebral edema, possibly due to the release of destructive polymorphonuclear neutrophil azurophilic enzymes. Our objectives were to measure plasma azurophilic enzyme levels in children with diabetic ketoacidosis, to correlate plasma azurophilic enzyme levels with diabetic ketoacidosis severity, and to determine whether azurophilic enzymes disrupt the blood-brain barrier in vitro. DESIGN: Prospective clinical and laboratory study. SETTING: The Children's Hospital, London Health Sciences Centre. SUBJECTS: Pediatric type 1 diabetes patients; acute diabetic ketoacidosis or age-/sex-matched insulin-controlled. MEASUREMENTS AND MAIN RESULTS: Acute diabetic ketoacidosis in children was associated with elevated polymorphonuclear neutrophils. Plasma azurophilic enzymes were elevated in diabetic ketoacidosis patients, including human leukocyte elastase (p < 0.001), proteinase-3 (p < 0.01), and myeloperoxidase (p < 0.001). A leukocyte origin of human leukocyte elastase and proteinase-3 in diabetic ketoacidosis was confirmed with buffy coat quantitative real-time polymerase chain reaction (p < 0.01). Of the three azurophilic enzymes elevated, only proteinase-3 levels correlated with diabetic ketoacidosis severity (p = 0.002). Recombinant proteinase-3 applied to human brain microvascular endothelial cells degraded both the tight junction protein occludin (p < 0.05) and the adherens junction protein VE-cadherin (p < 0.05). Permeability of human brain microvascular endothelial cell monolayers was increased by recombinant proteinase-3 application (p = 0.010). CONCLUSIONS: Our results indicate that diabetic ketoacidosis is associated with systemic polymorphonuclear neutrophil activation and degranulation. Of all the polymorphonuclear neutrophil azurophilic enzymes examined, only proteinase-3 correlated with diabetic ketoacidosis severity and potently degraded the blood-brain barrier in vitro. Proteinase-3 might mediate vasogenic edema during diabetic ketoacidosis, and selective proteinase-3 antagonists may offer future vascular- and neuroprotection.

Background Carbon monoxide (CO) inhalation protects organ by reducing inflammation and cell death during transplantation processes in animal model. However, using CO in clinical transplantation is difficult due to its delivery in a controlled manner. A manganese-containing CO releasing molecules (CORM)-401 has recently been synthesized which can efficiently deliver 3 molar equivalents of CO. We report the ability of this anti-inflammatory CORM-401 to reduce ischemia reperfusion injury associated with prolonged cold storage of renal allografts obtained from donation after circulatory death in a porcine model of transplantation. Methods To stimulate donation after circulatory death condition, kidneys from large male Landrace pig were retrieved after 1 hour warm ischemia in situ by cross-clamping the renal pedicle. Procured kidneys, after a brief flushing with histidine-tryptophan-ketoglutarate solution were subjected to pulsatile perfusion at 4°C with University of Wisconsin solution for 4 hours and both kidneys were treated with either 200 μM CORM-401 or inactive CORM-401, respectively. Kidneys were then reperfused with normothermic isogeneic porcine blood through oxygenated pulsatile perfusion for 10 hours. Urine was collected, vascular flow was assessed during reperfusion and histopathology was assessed after 10 hours of reperfusion. Results We have found that CORM-401 administration reduced urinary protein excretion, attenuated kidney damage markers (kidney damage marker-1 and neutrophil gelatinase-associated lipocalin), and reduced ATN and dUTP nick end labeling staining in histopathologic sections. CORM-401 also prevented intrarenal hemorrhage and vascular clotting during reperfusion. Mechanistically, CORM-401 appeared to exert anti-inflammatory actions by suppressing Toll-like receptors 2, 4, and 6. Conclusions Carbon monoxide releasing molecules-401 provides renal protection after cold storage of kidneys and provides a novel clinically relevant ex vivo organ preservation strategy.

Background: Diabetic ketoacidosis (DKA) in children is associated with cerebrovascular-related complications. We recently reported that DKA facilitates leukocyte adherence to the brain microvascular endothelium. Adhered leukocytes can release enzymes that instigate vascular dysfunction. Our aims were to measure plasma levels of leukocyte-derived matrix metalloproteinases (MMPs) from DKA patients and to correlate plasma MMP concentrations with DKA severity. Methods: Plasma was obtained from children with type 1 diabetes, either in DKA (n = 16) or insulin controlled (CON; n = 16). Antibody microarray and gelatin zymography were used to quantify plasma MMPs and their endogenous tissue inhibitors (TIMPs). MMP concentrations were correlated with DKA severity (blood pH). Quantitative PCR of leukocyte mRNA was used to help determine the origin of plasma MMPs. Results: DKA was associated with altered plasma levels of ↓MMP-2 (P < 0.001), ↑MMP-8 (P < 0.001), ↑MMP-9 (P < 0.05), and ↑TIMP-4 (P < 0.001), as compared with CON. Elevated MMP-8 and MMP-9 were both positively correlated with DKA severity (P < 0.05). DKA was associated with increased leukocyte mRNA for MMP-8, MMP-9, and TIMP-4 (P < 0.005). Conclusion: MMPs are dynamically regulated during DKA. Plasma MMP-8 and MMP-9 concentrations correlate with DKA severity and are known to degrade brain microvascular endothelial cell tight junctions. Thus, leukocyte-derived MMPs might contribute to DKA-associated cerebrovascular complications.

Polymorphonuclear leukocyte (PMN)-derived myeloperoxidase (MPO) contributes to the pathophysiology of numerous systemic inflammatory disorders through: (1) direct peroxidation of targets and (2) production of strong oxidizing compounds, e.g., hypohalous acids, particularly hypochlorous acid, which furthers oxidant damage and contributes to the propagation of inflammation and tissue injury/dysfunction. Carbon monoxide-releasing molecules (CORMs) offer potent anti-inflammatory effects; however, the mechanism(s) of action is not fully understood. This study assessed the potential of MPO activity inhibition by a water-soluble CORM, CORM-3. To this end, we used in vitro assays to study CORM-3-dependent modulation of MPO activity with respect to: (1) the inhibition of MPO's catalytic activity generally and (2) the specific inhibition of MPO's peroxidation and halogenation (i.e., production of hypochlorous acid) reactions. Further, we employed primary human umbilical vein endothelial cells (HUVECs) to investigate MPO-dependent cellular activation and dysfunction by measuring intracellular oxidant stress (DHR-123 oxidation) and HUVEC permeability (flux of Texas red-dextran), respectively. The results indicate that CORM-3 significantly inhibits MPO activity as well as MPO's peroxidation and hypohalous acid cycles specifically (p

Diabetic ketoacidosis (DKA) in children is associated with intracranial vascular complications, possibly due to leukocyte-endothelial interactions. Our aim was to determine whether DKA-induced inflammation promoted leukocyte adhesion to activated human cerebrovascular endothelium. Plasma was obtained from children with type 1 diabetes either in acute DKA or in an insulin-controlled state (CON). Plasma concentrations of 21 inflammatory analytes were compared between groups. DKA was associated with altered circulating levels of ↑CXCL1 (GROα), ↑CXCL8 (IL-8), ↑IL-6, ↑IFNα2, and ↓CXCL10 (IP-10) compared with CON. These plasma analyte measurements were then used to create physiologically relevant cytokine mixtures (CM). Human cerebral microvascular endothelial cells (hCMEC/D3) were stimulated with either plasma (DKA-P or CON-P) or CM (DKA-CM or CON-CM) and assessed for polymorphonuclear leukocyte (PMN) adhesion. Stimulation of hCMEC/D3 with DKA-P or DKA-CM increased PMN adhesion to hCMEC/D3 under “flow” conditions. PMN adhesion to hCMEC/D3 was suppressed with neutralizing antibodies to CXCL1/CXCL8 or their hCMEC/D3 receptors CXCR1/CXCR2. DKA-P, but not DKA-CM, initiated oxidative stress in hCMEC/D3. Expression of ICAM-1, VCAM-1, and E-selectin were unaltered on hCMEC/D3 by either DKA-P or DKA-CM. In summary, DKA elicits inflammation in children associated with changes in circulating cytokines/chemokines. Increased CXCL1/CXCL8 instigated PMN adhesion to hCMEC/D3, possibly contributing to DKA-associated intracranial vascular complications.

Carbon monoxide-releasing molecules (CORMs) suppress inflammation by reducing polymorphonuclear leukocyte (PMN) recruitment to the affected organs. We investigated modulation of PMN-endothelial cell adhesive interactions by water-soluble CORM-401 using an experimental model of endotoxemia in vitro. Human umbilical vein endothelial cells (HUVEC) grown on laminar-flow perfusion channels were stimulated with 1 μg/mL lipopolysaccharide for 6 hours and perfused with 100 μmol/L CORM-401 (or inactive compound iCORM-401)-pretreated PMN for 5 minutes in the presence of 1.0 dyn/cm HUVEC: PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMN with CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P < 0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distribution and fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P < 0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophil migratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics.

Background: Overwhelming systemic inflammation has been implicated in the progression of acute lung injury (ALI) leading to multiple organ failure (MOF) and death. Previous studies suggest that mechanical ventilation (MV) may be a key mediator of MOF through an upregulation of the systemic inflammatory response. Objectives: It was the aim of this study to investigate mechanisms whereby mechanical stress induced by different tidal volumes may contribute to the development of systemic inflammation and maladaptive peripheral organ responses in the setting of ALI. Methods: An acid aspiration model of ALI was employed in 129X1/SVJ mice through an intratracheal administration of hydrochloric acid followed by MV employing either a low (5 ml/kg) or high (12.5 ml/kg) tidal volume ventilation for 120 min. The isolated perfused mouse lung setup was used to assess the specific contribution of the lung to systemic inflammation during MV. Furthermore, lung perfusate collected over the course of MV was used to assess the effects of lung-derived mediators on activation (expression of a proadhesive phenotype) of liver endothelial cells. Results: High tidal volume MV of acid-injured lungs resulted in greater physiologic and histological indices of lung injury compared to control groups. Additionally, there was an immediate and significant release of multiple inflammatory mediators from the lung into the systemic circulation which resulted in greater levels of mRNA adhesion molecule expression in liver endothelial cells in vitro. Conclusions: This study suggests that MV, specifically tidal volume strategy, influences the development of MOF through an upregulation of lung-derived systemic inflammation resulting in maladaptive cellular changes in peripheral organs.

The parathyroid hormone receptor type 1 (PTHR1) is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP) and primarily signals via intracellular pathways involving adenylyl cyclase and phospholipase C. The intracellular tail domain of the PTHR1 contributes to G protein subunit coupling that is important for second messenger signalling. In addition, the intracellular domain has a potential nuclear localization sequence (NLS) that, if functional, could point to an intracrine role for the receptor. In the present study, we have utilized 2 sets of constructs that employ either a [KRK(484-486)AAA](3Ala) mutation in the putative NLS or the non-mutant counterpart and included (a) the full-length rat PTHR1 with FLAG and c-myc epitope tags at the N-terminus and C-terminus, respectively (designated as PTHR1(3Ala)-TAG and PTHR1-TAG); and (b) only the putative NLS-containing intracellular domain (471-488), with green fluorescent protein (GFP) fused to the C-terminus (designated as GFP-(3Ala)471-488 or GFP-471-488). Porcine kidney LLC-PK1 cells stably expressing the PTHR1(3Ala)-TAG exhibited reduced signalling via both cAMP and cytosolic calcium transients in spite of greater cell surface expression relative to cells expressing PTHR1-TAG. We also examined the ability of the intracellular tail to influence the cellular localization of a heterologous protein. LLC-PK1 cells transiently transfected with GFP-471-488, exhibited increased fluorescence within the nucleus, relative to cells transfected with GFP alone that was not observed when cells were transiently transfected with the mutated construct, GFP-(3Ala)471-488. However, LLC-PK1 cells transiently transfected with either the full-length PTHR1-TAG or the PTHR1(3Ala)-TAG constructs did not exhibit nuclear localization of these receptors. Moreover, mouse osteoblast-like cells (MC3T3-E1) transiently expressing PTHR1-TAG also failed to demonstrate nuclear localization, although both full-length PTHR1 constructs exhibited plasma membrane immunofluorescence in both cell lines. Thus, the 484-486 sequence is critical for the full signalling responsiveness of the intact PTHR1, but the putative nuclear localization signal may not function as such within the intact receptor.

Objective Exogenously administered CO interferes with PMN recruitment to the inflamed organs. The mechanisms of CO-dependent modulation of vascular proadhesive phenotype, a key step in PMN recruitment, are unclear. Methods We assessed the effects/mechanisms of CO liberated from a water-soluble CORM-3 on modulation of the proadhesive phenotype in hCMEC/D3 in an in vitro model of endotoxemia. To this end, hCMEC/D3 were stimulated with LPS (1 μg/mL) for six hours. In some experiments hCMEC/D3 were pretreated with CORM-3 (200 μmol/L) before LPS-stimulation. PMN rolling/adhesion to hCMEC/D3 were assessed under conditions of laminar shear stress (0.7 dyn/cm2). In parallel, expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 (qPCR), activation of transcription factors, NF-κB and AP-1 (ELISA), and MAPK-signaling (expression/phosphorylation of p38, ERK1/2, and JNK1/2; western blot) were assessed. Results The obtained results indicate that CORM-3 pretreatment reduces PMN rolling/adhesion to LPS-stimulated hCMEC/D3 (p

Previous work has demonstrated that carbon monoxide releasing molecules (CORMs) suppress inflammation, however, the mechanisms are not well understood. Neutrophil (PMN)-derived myeloperoxidase (MPO...

Objective To determine if the DKA-induced inflammation in juvenile mice provokes activation and dysfunction of CVECs. Methods DKA in juvenile mice was induced with administration of STZ and ALX. Blood from DKA mice was assessed for cytokines and soluble cell adhesion proteins, and either DKA plasma or exogenous compounds were applied to immortalized bEND3. Results DKA increased circulating levels of IL-6, IL-8(KC), MCP-1, IL-10, sE-selectin, sICAM-1, and sVCAM-1. Stimulation of bEND3 with DKA plasma caused cellular activation (increased ROS and activation of NF-κΒ), upregulation of a proadhesive phenotype (E-selectin, ICAM-1, and VCAM-1), and increased leukocyte-bEND3 interaction (leukocyte rolling/adhesion). TEER, a measure of bEND3 monolayer integrity, was decreased by DKA plasma. Activation and dysfunction of bEND3 with DKA plasma were suppressed by plasma heat treatment (56°C, 1 hour) and replicated with the application of DKA recombinant cytomix (IL-6, IL-8[KC], MCP-1, and IL-10), implicating circulating inflammatory protein(s) as mediators. Treatment of bEND3 with β-OH-butyrate, the main ketone elevated in DKA, failed to mimic the DKA plasma–induced activation and dysfunction of bEND3. Conclusions DKA elicits systemic inflammation associated with CVEC activation and dysfunction, possibly contributing to DKA-associated intracranial microvascular complications.

In the setting of acute lung injury, levels of circulating inflammatory mediators have been correlated with adverse outcomes. Previous studies have demonstrated that injured, mechanically ventilated lungs represent the origin of the host inflammatory response; however, mechanisms which perpetuate systemic inflammation remain uncharacterized. We hypothesized that lung-derived mediators generated by mechanical ventilation (MV) are amplified by peripheral organs in a “feed forward” mechanism of systemic inflammation. Herein, lung-derived mediators were collected from 129X1/SVJ mice after 2 hours of MV while connected to the isolated perfused mouse lung model setup. Exposure of liver endothelial cells to lung-derived mediators resulted in a significant increase in G-CSF, IL-6, CXCL-1, CXCL-2, and MCP-1 production compared to noncirculated control perfusate media (P<0.05). Furthermore, inhibition of the NF-κB pathway significantly mitigated this response. Changes in gene transcription were confirmed using qPCR for IL-6, CXCL-1, and CXCL-2. Additionally, liver tissue obtained from mice subjected to 2 hours ofin vivoMV demonstrated significant increases in hepatic gene transcription of IL-6, CXCL-1, and CXCL-2 compared to nonventilated controls. Collectively, this data demonstrates that lung-derived mediators, generated in the setting of MV, are amplified by downstream organs in a feed forward mechanism of systemic inflammation.