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Sepsis-elicited immunosuppression elevates the risk of secondary infections. We used a clinically relevant mouse model and serial peripheral blood samples from patients to assess the antimicrobial activities of mucosa-associated invariant T (MAIT) cells in sepsis. Hepatic and splenic MAIT cells from B6-MAIT

Coronavirus disease 2019 (COVID-19) is a systemic inflammatory condition with high mortality that may benefit from personalized medicine and high-precision approaches. COVID-19 patient plasma was analysed with targeted proteomics of 1161 proteins. Patients were monitored from Days 1 to 10 of their intensive care unit (ICU) stay. Age- and gender-matched COVID-19-negative sepsis ICU patients and healthy subjects were examined as controls. Proteomic data were resolved using both cell-specific annotation and deep-analysis for functional enrichment. COVID-19 caused extensive remodelling of the plasma microenvironment associated with a relative immunosuppressive milieu between ICU Days 3-7, and characterized by extensive organ damage. COVID-19 resulted in (1) reduced antigen presentation and B/T-cell function, (2) increased repurposed neutrophils and M1-type macrophages, (3) relatively immature or disrupted endothelia and fibroblasts with a defined secretome, and (4) reactive myeloid lines. Extracellular matrix changes identified in COVID-19 plasma could represent impaired immune cell homing and programmed cell death. The major functional modules disrupted in COVID-19 were exaggerated in patients with fatal outcome. Taken together, these findings provide systems-level insight into the mechanisms of COVID-19 inflammation and identify potential prognostic biomarkers. Therapeutic strategies could be tailored to the immune response of severely ill patients.

Aims Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aims to determine the underlying mechanisms, and to inform prognosis and treatment. Methods Plasma proteome from Long-COVID outpatients was analyzed in comparison to acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of approximately 3000 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity. Results Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cells with a resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This resetting of cell phenotypes was reflected in vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Levels of ANGPT1 and VEGFA were validated by serological methods in different patient cohorts. Silent signaling of transforming growth factor-β1 with elevated EP300 favored not only vascular inflammation, but also tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway was predicted that progressed from COVID-19 to Long-COVID. The vasculo-proliferative process identified in Long-COVID was associated with significant changes in the organ-specific proteome reflective of neurological and cardiometabolic dysfunction. Conclusions Taken together, our study uncovered a vasculo-proliferative process in Long-COVID initiated by prior hypoxia, and identified potential organ-specific prognostic biomarkers and therapeutic targets.

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.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

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.

Despite decades of preclinical research, no experimentally derived therapies for sepsis have been successfully adopted into routine clinical practice. Factors that contribute to this crisis of translation include poor representation by preclinical models of the complex human condition of sepsis, bias in preclinical studies, as well as limitations of single-laboratory methodology. To overcome some of these shortcomings, multicentre preclinical studies—defined as a research experiment conducted in two or more research laboratories with a common protocol and analysis—are expected to maximize transparency, improve reproducibility, and enhance generalizability. The ultimate objective is to increase the efficiency and efficacy of bench-to-bedside translation for preclinical sepsis research and improve outcomes for patients with life-threatening infection. To this end, we organized the first meeting of the National Preclinical Sepsis Platform (NPSP). This multicentre preclinical research collaboration of Canadian sepsis researchers and stakeholders was established to study the pathophysiology of sepsis and accelerate movement of promising therapeutics into early phase clinical trials. Integrated knowledge translation and shared decision-making were emphasized to ensure the goals of the platform align with clinical researchers and patient partners. 29 participants from 10 independent labs attended and discussed four main topics: (1) objectives of the platform; (2) animal models of sepsis; (3) multicentre methodology and (4) outcomes for evaluation. A PIRO model (predisposition, insult, response, organ dysfunction) for experimental design was proposed to strengthen linkages with interdisciplinary researchers and key stakeholders. This platform represents an important resource for maximizing translational impact of preclinical sepsis research.

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: 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

OBJECTIVES Acute limb compartment syndrome, a complication of musculoskeletal trauma, results in muscle necrosis and cell death. Carbon monoxide, liberated from the carbon monoxide-releasing molecule-3, has been shown protective in a rat model of compartment syndrome. The purpose of this study was to test the effect of carbon monoxide-releasing molecule-3 in a preclinical large animal model of compartment syndrome, with the ultimate goal of developing a pharmacologic adjunct treatment for compartment syndrome. DESIGN Animal research study. SETTING Basic research laboratory in a hospital setting. SUBJECTS Male Yorkshire-Landrace pigs (50-60 kg). INTERVENTIONS Pigs underwent 6 hours of intracompartmental pressure elevation by infusing fluid into the anterior compartment of the right hind limb. Carbon monoxide-releasing molecule-3 was administered systemically (2 mg/kg, IV) at fasciotomy, followed by 3-hour reperfusion. MEASUREMENTS AND MAIN RESULTS Muscle perfusion, inflammation, injury, and apoptosis were assessed in the skeletal muscle. Systemic leukocyte activation was assessed during compartment syndrome and reperfusion. Elevation of hind limb intracompartmental pressure resulted in significant microvascular perfusion deficits (44% ± 1% continuously perfused capillaries in compartment syndrome vs 76% ± 4% in sham; p < 0.001), increased tissue injury (ethidium bromide/bisbenzimide of 0.31 ± 0.07 in compartment syndrome vs 0.17 ± 0.03 in sham; p < 0.05), apoptosis (fluorescence in vivo/bisbenzimide of 0.26 ± 0.06 in compartment syndrome vs 0.13 ± 0.03 in sham; p < 0.05), and systemic leukocyte activation (14.7 relative luminescence units/10 polymorphonuclear leukocytes in compartment syndrome vs 1.0 ± 0.1 in baseline; p < 0.001). Systemic application of carbon monoxide-releasing molecule-3 at fasciotomy increased the number of continuously perfused capillaries (68% ± 3%; p < 0.001), diminished tissue injury (ethidium bromide/bisbenzimide of 0.13 ± 0.04; p < 0.05), apoptosis (fluorescence in vivo/bisbenzimide of 0.12 ± 0.03; p < 0.05), and blocked systemic leukocyte activation (3.9 ± 0.3 relative luminescence unit/10 polymorphonuclear leukocytes; p < 0.001). CONCLUSIONS Administration of carbon monoxide-releasing molecule-3 at fasciotomy offered protection against compartment syndrome-induced microvascular perfusion deficit, tissue injury, and systemic leukocyte activation. The data suggest the potential therapeutic application of carbon monoxide-releasing molecule-3 to patients at risk of developing compartment syndrome.

Ischemia-reperfusion (IR) is a limb- and life-threatening complication of acute limb ischemia and musculoskeletal trauma. Carbon monoxide-releasing molecules (CORMs) have recently been shown to protect microvascular perfusion and to reduce inflammation and injury in various ischemic animal models. The purpose of this study was to examine the effects of water-soluble CORM-3 on the extent of IR-induced muscle injury.Wistar rats were randomized into three groups: sham (no ischemia), IR + CORM-3 (10 mg/kg intraperitoneally), and IR + inactive CORM-3 (iCORM-3; 10 mg/kg intraperitoneally). No-flow ischemia was induced by the application of a tourniquet to the hind limb for 2 hours; tourniquet release commenced the reperfusion phase. Both CORM-3 and iCORM-3 were injected immediately after tourniquet release. Temporal changes in microvascular perfusion, cellular tissue injury (ethidium bromide and bisbenzimide staining), and inflammatory response (leukocyte recruitment) within the extensor digitorum longus muscle were assessed using intravital video microscopy every 15 minutes for a total of 90 minutes after initiation of reperfusion. Systemic levels of tumor necrosis factor-α were also measured.Hind limb IR resulted in (1) a significant no-reflow phenomenon followed by progressive increase in microvascular perfusion deficit (21% ± 2% continuously perfused capillaries in IR vs 76% ± 4% in sham [P .001]; 52% ± 8% nonperfused capillaries in IR vs 13% ± 2% in sham at 90 minutes of reperfusion [P .001]), (2) tissue injury (ethidium bromide and bisbenzimide staining of 0.52 ± 0.07 in IR vs 0.05 ± 0.03 in sham at 90 minutes of reperfusion [P .001]), (3) leukocyte recruitment (13.7 ± 0.9 adherent leukocytes/30 seconds/1000 μmCORM-3 displays potent protective and anti-inflammatory effects in an experimental model of hind limb IR, suggesting a potential therapeutic application of CORMs in treatment of ischemic conditions.

Alveolar macrophages are key contributors to both the promotion and resolution of inflammation in the lung and are categorized into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. The change in M1/M2 balance has been reported in various pulmonary diseases and is a target for therapeutic intervention. The aim of this study was to assess the modulation of M1/M2 phenotype in alveolar macrophages by water-soluble carbon monoxide-releasing molecule-3 (CORM-3). Rat alveolar macrophages (AM) (NR8383) in culture were stimulated with LPS (5 ng/ml)/IFN-γ (10 U/ml) or IL-4 (10 ng/ml)/IL-13 (10 ng/ml) to induce M1 and M2 phenotypes, respectively. Expression of M1 phenotype markers, iNOS and TNF-α, and M2 phenotype markers, CD206 and Ym-1, was assessed by western blotting after 1, 3, 6, or 24 h in the absence or presence of CORM-3 (0.15 mM) treatment. Inactive CORM-3 (iCORM-3) was used as a control. Treatment of naïve (unstimulated) AM with CORM-3 promoted progression of the M2 phenotype as evidenced by the increased expression of CD206 (at 1 h; 1.8-fold) and Ym-1 (at 3 h; 1.9-fold), respectively. Surprisingly, CORM-3 treatment also upregulated the expression of iNOS protein as assessed 6 h following stimulation of AM with CORM-3 (2.6-fold). On the contrary, CORM-3 effectively reduced LPS/IFN-γ-induced expression of iNOS protein (0.6-fold); however, it had no effect on TNF-α expression. Finally, CORM-3 acutely (1-3 h) upregulated CD206 (1.4-fold) and Ym-1 (1.6-fold) levels in IL-4-/IL-13-treated (M2-stimulus) macrophages. These findings indicate that CORM-3 modulates macrophage M1 and M2 phenotypes in vitro with respect to continuous suppression of iNOS expression in M1-polarized macrophages and transient (early-phase) upregulation of CD206 and Ym-1 proteins in M2-polarized macrophages.

© 2019 John Wiley & Sons Ltd Objective: Limb compartment syndrome (CS), a complication of trauma, results in muscle necrosis and cell death; ischemia and inflammation contribute to microvascular dysfunction and parenchymal injury. Carbon monoxide-releasing molecule-3 (CORM-3) has been shown to protect microvascular perfusion and reduce inflammation in animal models of CS. The purpose of the study was to test the effect of CORM-3 in human in vitro CS model, allowing exploration of the mechanism(s) of CO protection and potential development of pharmacologic treatment. Methods: Confluent human vascular endothelial cells (HUVECs) were stimulated for 6 h with serum isolated from patients with CS. Intracellular oxidative stress (production of reactive oxygen species (ROS)) apoptosis, transendothelial resistance (TEER), polymorphonuclear leukocyte (PMN) activation and transmigration across the monolayer in response to the CS stimulus were assessed. All experiments were performed in the presence of CORM-3 (100 μM) or its inactive form, iCORM-3. Results: CS serum induced a significant increase in ROS, apoptosis and endothelial monolayer breakdown; it also increased PMN superoxide production, leukocyte rolling and adhesion/transmigration. CORM-3 completely prevented CS-induced ROS production, apoptosis, PMN adhesion, rolling and transmigration, while improving monolayer integrity. Conclusion: CORM-3 offers potent anti-oxidant and anti-inflammatory effects, and may have a potential application to patients at risk of developing CS.

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.

OBJECTIVES To examine the protective effects of carbon monoxide (CO), liberated from a novel CO-releasing molecule (CORM-3), on the function of compartment syndrome (CS)-challenged muscle in a rodent model, thus providing for a potential development of a pharmacologic adjunctive treatment for CS. METHODS Wistar rats were randomized into 4 groups: sham (no CS), CS, CS with inactive CORM-3 (iCORM-3), and CS + CORM-3 (10 mg/kg intraperitoneally). CS was induced by elevation of intracompartmental pressure to 30 mm Hg through an infusion of isotonic saline into the anterior compartment of the hind limb for 2 hours. Both CORM-3 and iCORM-3 were injected immediately after fasciotomy. Microvascular perfusion, cellular tissue injury, and inflammatory response within the extensor digitorum longus muscle were assessed using intravital video microscopy 45 minutes after fasciotomy. Systemic levels of tumor necrosis factor alpha (TNF-α) were also measured. RESULTS Elevation of intracompartmental pressure resulted in significant microvascular perfusion deficits (23% ± 2% continuously perfused capillaries in CS vs. 76% ± 4% in sham, P < 0.0001; 55% ± 2% nonperfused capillaries in CS vs. 13% ± 2% in sham, P < 0.0001), significant increase in tissue injury (ethidium bromide/bisbenzimide of 0.31 ± 0.05 in CS vs. 0.05 ± 0.03 in sham, P < 0.0001) and adherent leukocytes (13.7 ± 0.9 in CS vs. 1.8 ± 0.5 in sham, P < 0.0001), and a progressive rise in systemic TNF-α. CORM-3 (but not iCORM-3) treatment restored the number of continuously perfused capillaries (57% ± 5%, P < 0.001), diminished tissue injury (ethidium bromide/bisbenzimide of 0.07 ± 0.01, P < 0.001), reversed the CS-associated rise in TNF-α, and decreased leukocyte adherence (0.6 ± 0.3, P < 0.001). CONCLUSIONS CORM-3 displays a potent protective/anti-inflammatory action in an experimental model of CS, suggesting a potential therapeutic application to patients at risk of developing CS.

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.

Organ cold storage and subsequent transplantation are associated with significant ischemia-reperfusion injury, leading to cell death, graft inflammation and decreased graft function.CORM-3s reduce oxidative stress and prevent inflammation in kidneys stored at 4C and subsequently transplanted. Graft survival and function are markedly improved compared to kidneys preserved and stored in University of Wisconsin solution alone. We determined whether CORM-3 has direct antiapoptotic effects on in vitro preparations of human HUVECs exposed to anoxic conditions. We also determined whether direct administration of CORM-3 to renal grafts before and/or after cold storage would prevent renal damage during the transplantation process.CORM-3 supplementation led to a significantly increased frequency of live cells (mean ± SD 72.3% ± 1.9%, p0.01), reduced apoptosis (14.9% ± 6.1%, p0.01) and decreased mitochondrial transmembrane potential (40.2% ± 7.2%, p0.05) in HUVECs exposed to 20 hours of cold storage compared to controls (11.6% ± 3.5%, 82.2% ± 2.3% and 78.2% ± 3.2%, respectively). In keeping with this antiapoptotic effect CORM-3 supplementation led to a mean 7.4 ± 2.1-fold up-regulation in Bcl-2 gene expression. CORM-3 supplementation in standard preservation solution was most beneficial at initial ischemic injury and before cold storage exposure. However, additional reflushing before vascular reperfusion showed an additive benefit to graft survival and function after transplantation. This was confirmed by decreased glomerular and tubular necrosis, and apoptosis in double flushed grafts.CORM-3 supplementation in standard University of Wisconsin solution has a significant impact on decreasing cellular and graft injury, and improving survival through its antiapoptotic effects, which are likely mediated through mitochondrial membrane stabilization.

Translational research often involves tissue sampling and analysis. Blood is by far the most common tissue collected. Due to the many difficulties encountered with blood procurement from children, it is imperative to maximize the quality and stability of the collected samples to optimize research results. Collected blood can remain whole or be fractionated into serum, plasma, or cell concentrates such as red blood cells, leukocytes, or platelets. Serum and plasma can be used for analyte studies, including proteins, lipids, and small molecules, and as a source of cell-free nucleic acids. Cell concentrates are used in functional studies, flow cytometry, culture experiments, or as a source for cellular nucleic acids. Before initiating studies on blood, a thorough evaluation of practices that may influence analyte and/or cellular integrity is required. Thus, it is imperative that child health researchers working with human blood are aware of how experimental results can be altered by blood sampling methods, times to processing, container tubes, presence or absence of additives, shipping and storage variables, and freeze-thaw cycles. The authors of this review, in an effort to encourage and optimize translational research using blood from pediatric patients, outline best practices for blood collection, processing, shipment, and storage.

The brain of the infant and young child is a developing, dynamic, structure subject to functional remodelling under the influence of factors responsible for optimal neuronal development and synaptogenesis. It exhibits age dependent variation in metabolic rate, blood flow, and ability to tolerate oxidative stress. It is also characterized by an exuberance of neurotransmitter activity, particularly in the first few years of life. The dynamic evolution and adaptability of early brain function permits the organization of neuronal networks to be influenced by environmental stimulation, and, to reduce the functional impact of injury. However, these same processes may also exacerbate the harm sustained by the brain following an acquired brain injury (ABI). The developing neurons are susceptible to excitotoxicity, oxidative stress, and, inflammation, often leading to cellular necrosis and apoptosis. Despite being immunologically privileged via the blood brain barrier, the developing brain is susceptible to injury from systemic inflammation through alteration of normally protective cerebrovascular endothelial cell function. Finally, many of the therapeutic agents currently employed in post-ABI hospital care may also compromise ABI outcome via non-intended pharmacological effects. These agents include analgesic, sedative and anti-convulsant medications. This review emphasizes those physiological considerations in the developing brain which may impact the outcome after ABI, including, the cellular mechanisms of neuronal and cerebrovascular endothelial cell injury, ABI outcome and future therapeutic directions.

The NLRP3 inflammasome is an intracellular multiple-protein complex that controls the maturation and release of interleukin (IL)-1β and IL-18. Endogenous carbon monoxide (CO) is anti-inflammatory. The aim of this study was to assess the effects/mechanisms of CO-releasing molecule-3 (CORM-3)-dependent modulation of the NLRP3 inflammasome in cardiac fibroblasts (CF) and its effect on myocardial function in sepsis. CF were treated with CORM-3 or inactive CORM-3 (iCORM-3) before NLRP3 inflammasome priming with lipopolysaccharides (LPS) or following activation with adenosine triphosphate (ATP). In parallel, cardiomyocytes (CM) were challenged with supernatants of LPS/ATP-stimulated CF or a cytokine mixture (Cyto-mix) containing IL-1β, IL-18, and HMGB1. In vivo, mice were treated with CORM-3 before or after LPS to induce sepsis (endotoxemia). Pretreatment of CF with CORM-3 prevented an LPS-induced increase in NLRP3 and pro-IL-1β expression. Treatment of CF with CORM-3 before ATP prevented ATP-induced activation of the NLRP3 inflammasome. Challenging CF with LPS/ATP promoted NLRP3 interactions with adaptor ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), which was prevented by CORM-3. Challenging CM with supernatants of CF with LPS/ATP or Cyto-mix (IL-1β, IL-18, and HMGB1) resulted in CM apoptosis, which was attenuated with either a CORM-3 or IL-1 receptor antagonist. Finally, myocardial NLRP3 inflammasome activation and myocardial dysfunction in septic mice were abolished by CORM-3. In NLRP3-deficient mice with sepsis, CORM-3 did not show additional benefits in improving myocardial function. Our results indicate that CORM-3 suppresses NLRP3 inflammasome activation by blocking NLRP3 interactions with the adaptor protein ASC and attenuates myocardial dysfunction in mice with sepsis.

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.

INTRODUCTION AND OBJECTIVES: Definitive treatment of symptomatic post transplant lymphocele includes percutaneous aspiration with or without sclerotherapy, or open/laparoscopic transperitoneal lymphocele drainage. Some authors have demonstrated that lymphocele formation can virtually be prevented by placing an intraoperative drain.We herein prospectively investigated the effect of placing a retroperitoneal drain during renal transplantation surgery on the incidence of lymphocele formation in recipients compared to those who received no drain. METHODS: a totalof315adult patientswhounderwent living renal transplantations in our institution were prospectively enrolled. The patient were randomlyassigned to oneof two groupson thebasis ofwhether or not they received a Jackson Pratt drain in the retroperitoneal space during surgery; group 1 had a drain placed (n1⁄4203) and group 2 did not (n1⁄4112). Patient demographic and clinical data known to affect transplant outcomes were compared between group 1 (drain) and group 2 ( no drain), including recipient age, gender, bodymass index (BMI) at transplantation, cause of end-stage renal disease, dialysis before transplantation, donor age, anastomotic time, delayed graft function and acute rejection episodes. This was correlated with lymphocele incidence rate and the need for lymphocele intervention (operative or percutaneous radiological). RESULTS: Individual risk factors were analyzed for their association with lymphocele formation in a univariate model. Themost important factors associated with an increased risk of lymphocele formation were no drain (P1⁄40.002), high BMI (p1⁄40.003) and diabetic nephropathy as an underlying cause of end stage renal failure (0.0426). The presence of diabetic nephropathy and anastomotic timewasassociatedwith increased risk but was not significant (P 1⁄40.264 and 0.690, respectively). CONCLUSIONS: In conclusion, placing of a retroperitoneal drain during renal transplant surgery significantly decreases the incidence of lymphocele formation. Furthermore, it significantly reduces the need for surgical intervention as a treatment modality for clinically significant lymphoceles. We recommend routine placement of a retroperitoneal drain during kidney transplant surgery in all patients.

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.

A compelling strategy for treatment of spinal cord injury is the blockade of integrin-mediated leukocyte extravasation using a monoclonal antibody (mAb) against the alpha4 subunit of the alpha4beta1-integrin. However, little is known with respect to neutrophil function following anti-alpha4 mAb treatment. This study assessed the effects of anti-alpha4 mAb binding on neutrophil activation [reactive oxygen species (ROS) production], function (phagocytic activity) and anti-alpha4-mAb/alpha4beta1-integrin-complex internalization.Resting, primed or stimulated rat neutrophils were incubated ex vivo with anti-alpha4 mAb or isotype-control antibody. ROS production, phagocytic activity, and anti-alpha4-mAb/alpha4beta1-integrin-complex internalization were determined by flow cytometry using dihydrorhodamine (DHR1,2,3), fluorescent microspheres, and indirect immunolabeling, respectively.Brief (0.5 h) incubation of resting, primed or activated neutrophils with anti-alpha4 mAb had no effect on ROS production and did not change neutrophil phagocytic activity. However, prolonged incubation (2 h), assessed only in resting neutrophils, increased ROS production. The anti-alpha4-mAb/alpha4beta1-integrin-complex was internalized after 1 h of anti-alpha4 mAb treatment and remained internalized up to 6 h.Neutrophil ROS production and phagocytic function remain unaltered after brief anti-alpha4 mAb exposure, demonstrating that use of this mAb as a treatment should not adversely affect important beneficial roles of these cells.

CORM-released CO has been shown to be beneficial in resolution of acute inflammation. The acute phase of intestinal ischemia-reperfusion (I/R) injury is characterized by oxidative stress-related inflammation and leukocyte recruitment. In this study, we assessed the effects and potential mechanisms of CORM-2-released CO in modulation of inflammatory response in the small intestine following I/R-challenge. To this end mice (C57Bl/6) small intestine were challenged with ischemia by occluding superior mesenteric artery (SMA) for 45 min. CORM-2 (8 mg/kg; i.v.) was administered immediately before SMA occlusion. Sham operated mice were injected with vehicle (0.25% DMSO). Inflammatory response in the small intestine (jejunum) was assessed 4 h following reperfusion by measuring tissue levels of TNF-alpha protein (ELISA), adhesion molecules E-selectin and ICAM-1 (Western blot), NF-kappaB activation (EMSA), along with PMN tissue accumulation (MPO assay) and leukocyte rolling/adhesion in the microcirculation of jejunum (intravital microscopy). The obtained results indicate that tissue levels of TNF-alpha, E-selectin and ICAM-1 protein expression, activation of NF-kappaB, and subsequent accumulation of PMN were elevated in I/R-challenged jejunum. The above changes were significantly attenuated in CORM-2-treated mice. Taken together these findings indicate that CORM-2-released CO confers anti-inflammatory effects by interfering with NF-kappaB activation and subsequent up-regulation of vascular pro-adhesive phenotype in I/R-challenged small intestine.

Systemic inflammatory response syndrome, as a consequence of ischemia/reperfusion (I/R), negatively influences the function of the affected organs. The objective of this study was to assess the role of nitric oxide (NO) in remote intestinal inflammatory response elicited by hindlimb I/R. To this end, C57BL/6 (wild type; WT) and inducible nitric-oxide synthase (iNOS)-deficient mice were subjected to bilateral hindlimb ischemia (1 h) followed by 6 h of reperfusion. Some WT mice were injected with iNOS inhibitor N-[3-(aminomethyl)benzyl] acetamidine (1400W) (5 mg/kg s.c.) immediately before reperfusion, and proinflammatory response was assessed 6 h later. Hindlimb I/R resulted in dysfunction of the small intestine as assessed by the increase in permeability [blood-to-lumen clearance of Texas Red-dextran (molecular mass 3 kDa)] and an increase in the luminal levels of tumor necrosis factor (TNF)-alpha protein and nitrate/nitrite (NO(2)(-)/NO(3)(-)). The above-mentioned changes were accompanied by up-regulation of the proinflammatory phenotype in the mucosa of small intestine with respect to 1) an increase in TNF-alpha and iNOS protein expression, 2) leukocyte accumulation, 3) formation of edema, 4) an increase in leukocyte rolling/adhesion in the submucosal microvasculature, and 5) activation of transcription factor nuclear factor-kappaB and up-regulation of adhesion molecule expression. Interestingly, the most profound changes with respect to intestinal dysfunction were found in jejunum and ileum, whereas duodenum was affected the least. Interfering with iNOS activity (1400W and iNOS-deficient mice) significantly attenuated hindlimb I/R-induced inflammatory response and dysfunction of the small intestine with respect to the above-mentioned markers of inflammation. The obtained results indicate that hindlimb I/R induces remote inflammatory response in the small intestine through an iNOS-derived NO-dependent mechanism.

Sepsis-associated encephalopathy is an early manifestation of sepsis, resulting in a diffuse dysfunction of the brain. Recently, nitric oxide (NO) has been proposed to be one of the key molecules involved in the modulation of inflammatory responses in the brain. The aim of this study was to assess the role of NO in cerebrovascular endothelial cell activation/dysfunction during the early onsets of sepsis. To this end, we employed an in vitro model of sepsis in which cultured mouse cerebrovascular endothelial cells (MCVEC) were challenged with blood plasma (20% vol/vol) obtained from sham or septic (feces-induced peritonitis, FIP; 6 h) mice. Exposing MCVEC to FIP plasma for 1 h resulted in increased production of reactive oxygen species and NO as assessed by intracellular oxidation of oxidant-sensitive fluorochrome, dihydrorhodamine 123 (DHR 123), and nitrosation of NO-specific probe, DAF-FM, respectively. The latter events were accompanied by dissociation of tight junction protein, occludin, from MCVEC cytoskeletal framework and a subsequent increase in FITC-dextran (3-kDa mol mass) flux across MCVEC grown on the permeable cell culture supports, whereas Evans blue-BSA (65-kDa mol mass) or FITC-dextran (10-kDa mol mass) flux were not affected. FIP plasma-induced oxidant stress, occludin rearrangement, and MCVEC permeability were effectively attenuated by antioxidant, 1-pyrrolidinecarbodithioic acid (PDTC; 0.5 mM), or interfering with nitric oxide synthase (NOS) activity [0.1 mM nitro-l-arginine methyl ester (l-NAME) or endothelial NOS (eNOS)-deficient MCVEC]. However, treatment of MCVEC with PDTC failed to interfere with NO production, suggesting that septic plasma-induced oxidant stress in MCVEC is primarily a NO-dependent event. Taken together, these data indicate that during early sepsis, eNOS-derived NO exhibits proinflammatory characteristics and contributes to the activation and dysfunction of cerebrovascular endothelial cells.