Sepsis in surgical patients: Personalized medicine in the future treatment of sepsis.

Sepsis results when a severe infection overwhelms the normal regulatory mechanisms of the immune system, resulting in a dysregulated host response characterized by new-onset organ failure. A wide range of infectious challenges can induce sepsis, resulting in an even wider range of maladaptive immune responses. This makes sepsis a syndromic diagnosis without a unifying, underlying molecular mechanism. The next step toward personalized medicine for sepsis is to resolve the heterogeneity across the universe of septic patients in order to establish pathobiologically homogenous sepsis "endotypes" that have uniformly defined changes in physiology and immunology. Defining the mechanisms of immune dysfunction within these endotypes will provide a roadmap for the application of immunomodulatory therapies for sepsis. This approach can drive in a paradigm shift in sepsis treatment, moving beyond supportive care and toward active efforts to restore normal immune function.

Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

BACKGROUND: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis that the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. METHODS: Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. RESULTS: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. CONCLUSION: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Adverse outcomes and an immunosuppressed endotype in septic patients with reduced IFN-γ ELISpot.

BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.

Adverse Long-Term Outcomes and an Immune Suppressed Endotype in Sepsis Patients with Reduced Interferon-γELISpot: A Multicenter, Prospective Observational Study.

BACKGROUND: Sepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients has generally relied on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision. METHODS: An ex vivo whole blood enzyme-linked immunosorbent (ELISpot) assay for cellular production of interferon-γ (IFN-γ) was evaluated in 107 septic and 68 non-septic patients from five academic health centers using blood samples collected on days 1, 4 and 7 following ICU admission. RESULTS: Compared with 46 healthy subjects, unstimulated and stimulated whole blood IFNγ expression were either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole blood IFNγ expression was significantly reduced on ICU days 1, 4 and 7 (all p<0.05), due to both significant reductions in total number of IFNγ producing cells and amount of IFNγ produced per cell (all p<0.05). Importantly, IFNγ total expression on day 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6 and procalcitonin. Septic patients with low IFNγ expression were older and had lower ALC and higher sPD-L1 and IL-10 concentrations, consistent with an immune suppressed endotype. CONCLUSIONS: A whole blood IFNγ ELISpot assay can both identify septic patients at increased risk of late mortality, and identify immune-suppressed, sepsis patients.

Defects in vein valve PROX1/FOXC2 antithrombotic pathway in endothelial cells drive the hypercoagulable state induced by trauma and critical illness.

OBJECTIVES: Deep venous thrombosis (DVT) causes significant morbidity and mortality after trauma. Recently, we have shown that blood flow patterns at vein valves induce oscillatory stress genes, which maintain an anticoagulant endothelial phenotype that inhibits spontaneous clotting at vein valves and sinuses, is lost in the presence of DVT in human pathological samples, and is dependent on expression of the transcription factor FOXC2. We describe an assay, modifying our mouse multiple injury system, which shows evidence of clinically relevant microthrombosis and hypercoagulability applicable to the study of spontaneous DVT in trauma without requiring direct vascular injury or ligation. Finally, we investigated whether these model findings are relevant to a human model of critical illness by examining gene expression changes by quantitative polymerase chain reaction and immunofluorescence in veins collected from critically ill. METHODS: C57/Bl6 mice were subjected to a modified mouse multiple injury model with liver crush injury, crush and pseudofracture of a single lower extremity, and 15% total blood volume hemorrhage. Serum was assayed for d-dimer at 2, 6, 24, and 48 hours after injury by enzyme-linked immunosorbent assay. For the thrombin clotting assay, veins of the leg were exposed, 100 µL of 1 mM rhodamine (6 g) was injected retro-orbitally, and 450 µg/mL thrombin was then applied to the surface of the vein with examination of real-time clot formation via in vivo immunofluorescence microscopy. Images were then examined for percentage area of clot coverage of visible mouse saphenous and common femoral vein. Vein valve specific knockout of FOXC2 was induced with tamoxifen treatment in PROX1 Ert2Cre FOXC2 fl/fl mice as previously described. Animals were then subjected to a modified mouse multiple injury model with liver crush injury, crush and pseudofracture of a single lower extremity, and 15% total blood volume hemorrhage. Twenty-four hours after injury, we examined the valve phenotype in naive versus multiple injury animals, with and without loss of the FOXC2 gene from the vein valve (FOXC2 del ) via the thrombin assay. Images were then examined for proximity of clot formation to the valve present at the junction of the mouse saphenous, tibial, and superficial femoral vein and presence of spontaneous microthrombi present in the veins before exposure to thrombin. Human vein samples were obtained from excess tissue preserved after harvest for elective cardiac surgery and from organ donors after organ procurement. Sections were submitted for paraffin embedding and then assayed by immunofluorescence for PROX1, FOXC2, thrombomodulin, endothelial protein C receptor, and von Willebrand's factor. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee, and all human studies reviewed and approved by the institutional review board. RESULTS: After mouse multiple injuries, enzyme-linked immunosorbent assay for d-dimer showed evidence of products of fibrin breakdown consistent with formation of clot related to injury, fibrinolysis, and/or microthrombosis. The thrombin clotting assay demonstrated higher percentage area of vein covered with clot when exposed to thrombin in the multiple injury animals compared with uninjured (45% vs. 27% p = 0.0002) consistent with a phenotype of hypercoagulable state after trauma in our model system. Unmanipulated FoxC2 knockout mice manifest increased clotting at the vein valve as compared with unmanipulated wild type animals. After multiple injuries, wild type mice manifest increase clotting at the vein after thrombin exposure ( p = 0.0033), and equivalent to that of valvular knockout of FoxC2 (FoxC2del), recapitulating the phenotype seen in FoxC2 knockout animals. The combination of multiple injuries and FoxC2 knockout resulted in spontaneous microthrombi in 50% of the animals, a phenotype not observed with either multiple injuries or FoxC2 deficiency alone (χ 2 , p = 0.017). Finally, human vein samples demonstrated the protective vein valve phenotype of increased FOXC2 and PROX1 and showed decreased expression in the critically ill organ donor population by immunofluorescence imaging in organ donor samples. CONCLUSION: We have established a novel model of posttrauma hypercoagulation that does not require direct restriction of venous flow or direct injury to the vessel endothelium to assay for hypercoagulability and can generate spontaneous microthrombosis when combined with valve-specific FOXC2 knockout. We find that multiple injuries induce a procoagulant phenotype that recapitulates the valvular hypercoagulability seen in FOXC2 knockout and, in critically ill human specimens, find evidence for loss of oscillatory shear stress-induced gene expression of FOXC2 and PROX1 in the valvular endothelium consistent with potential loss of DVT-protective valvular phenotype.

IL-1ß expression in bone marrow dendritic cells is induced by TLR2 agonists and regulates HSC function.

Hematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1ß (IL-1ß) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1-/- mice show that TLR1/2-induced HSPC expansion is dependent on IL-1ß signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1ß and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1ß expression.

Cellular immunophenotype of major spine surgery in adults.

PURPOSE: ASD reconstructions are a major, sterile traumatic insult, likely causing perturbations to the immune systems. The immune response to surgery is associated with outcomes. The purpose of this study was to examine for a detectable immune signature associated with ASD surgery. METHODS: Consecutive patients undergoing ASD surgery were approached and enrolled. Peripheral blood was drawn before incision, 4 h after, and 24 h after incision. Blood was stabilized and comprehensive flow cytometric immunophenotyping performed. Leukocyte population frequency, absolute number and activation marker expression were defined. Immunologic features were defined and analyzed by hierarchical clustering and principal component analysis (PCA). Changes over time were evaluated by repeated measures ANOVA (RMANOVA) and were corrected for a 1% false discovery rate. Post hoc testing was by Dunn's test. p values of < = 0.05 were considered significant. RESULTS: Thirteen patients were enrolled; 11(85%) F, 65.4 years (± 7.5), surgical duration 418 ± 83 min, EBL 1928 ± 1253 mL. Hierarchical clustering and PCA found consistent time from incision-dependent changes. HLA-DR and activating co-stimulatory molecule CD86 were depressed at 4 h and furthermore at 24 h on monocyte surfaces. CD4 + HLA-DR + T cells, but not CD8 +, increased over time with increased expression of PD-1 at 4 and 24 h. CONCLUSIONS: Despite surgery and patient heterogeneity, we identified an immune signature associated with the sterile trauma of ASD surgery. Circulating leukocyte populations change in composition and signaling protein expression after incision and persisting to 24 h after incision, suggesting an immunocompromised state. Further work may determine relationships between this state and poor outcomes after surgery.

Dysregulation of the leukocyte signaling landscape during acute COVID-19.

The global COVID-19 pandemic has claimed the lives of more than 750,000 US citizens. Dysregulation of the immune system underlies the pathogenesis of COVID-19, with inflammation mediated tissue injury to the lung in the setting of suppressed systemic immune function. To define the molecular mechanisms of immune dysfunction in COVID-19 we utilized a systems immunology approach centered on the circulating leukocyte phosphoproteome measured by mass cytometry. We find that although COVID-19 is associated with wholesale activation of a broad set of signaling pathways across myeloid and lymphoid cell populations, STAT3 phosphorylation predominated in both monocytes and T cells. STAT3 phosphorylation was tightly correlated with circulating IL-6 levels and high levels of phospho-STAT3 was associated with decreased markers of myeloid cell maturation/activation and decreased ex-vivo T cell IFN-γ production, demonstrating that during COVID-19 dysregulated cellular activation is associated with suppression of immune effector cell function. Collectively, these data reconcile the systemic inflammatory response and functional immunosuppression induced by COVID-19 and suggest STAT3 signaling may be the central pathophysiologic mechanism driving immune dysfunction in COVID-19.

Interleukin-7 Reverses Lymphopenia and Improves T-Cell Function in Coronavirus Disease 2019 Patient With Inborn Error of Toll-Like Receptor 3: A Case Report.

BACKGROUND: Immunotherapy treatment for coronavirus disease 2019 combined with antiviral therapy and supportive care remains under intense investigation. However, the capacity to distinguish patients who would benefit from immunosuppressive or immune stimulatory therapies remains insufficient. Here, we present a patient with severe coronavirus disease 2019 with a defective immune response, treated successfully with interleukin-7 on compassionate basis with resultant improved adaptive immune function. CASE SUMMARY: A previously healthy 43-year-old male developed severe acute respiratory distress syndrome due to the severe acute respiratory syndrome coronavirus 2 virus with acute hypoxemic respiratory failure and persistent, profound lymphopenia. Functional analysis demonstrated depressed lymphocyte function and few antigen-specific T cells. Interleukin-7 administration resulted in reversal of lymphopenia and improved T-cell function. Respiratory function and clinical status rapidly improved, and he was discharged home. Whole exome sequencing identified a deleterious autosomal dominant mutation in TICAM1, associated with a dysfunctional type I interferon antiviral response with increased severity of coronavirus disease 2019 disease. CONCLUSIONS: Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening severe coronavirus disease 2019 infections, particularly by applying a precision medicine approach in selecting patients expressing an immunosuppressive phenotype.

IL-7 Immunotherapy in a Nonimmunocompromised Patient With Intractable Fungal Wound Sepsis.

A nonimmunocompromised patient developed life-threatening soft tissue infection with Trichosporon asahii, Fusarium, and Saksenaea that progressed despite maximum antifungal therapies and aggressive debridement. Interleukin-7 immunotherapy resulted in clinical improvement, fungal clearance, reversal of lymphopenia, and improved T-cell function. Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening fungal infections.

Pseudomonas aeruginosa Pneumonia Causes a Loss of Type-3 and an Increase in Type-1 Innate Lymphoid Cells in the Gut.

BACKGROUND: Sepsis induces gut barrier dysfunction characterized by increased gut epithelial apoptosis and increased intestinal permeability. The cytokine IL-22 has been demonstrated to regulate gut barrier function. Type-3 innate lymphoid cells (ILC3) are the predominate source of IL-22 in the GI tract. We hypothesized that sepsis may cause changes to the gut ILC3/IL-22 axis. MATERIALS AND METHODS: Sepsis was induced in WT and IL-22 KO mice by Pseudomonas aeruginosa pneumonia. Changes in gut-associated leukocyte populations were determined by flow-cytometry and ILC-associated transcripts were measured by RT-PCR. The effect of sepsis on gut permeability, pulmonary microbial burden, gut epithelial apoptosis, and survival was compared between WT and IL-22-/- mice. RESULTS: Sepsis resulted in a significant decrease in the number of ILC3 in the gut, with a reciprocal increase in type-1 ILC (ILC1). Consistent with prior reports, sepsis was associated with increased gut permeability; however there was no difference in gut permeability, gut epithelial apoptosis, pulmonary microbial burden, or survival between WT and IL-22-/- mice. CONCLUSIONS: Septic pneumonia causes a decrease in gut-associated ILC3 and an associated reciprocal increase in ILC1. This may reflect inflammation-induced conversion of ILC3 to ILC1. Constitutive systemic IL-22 deficiency does not alter sepsis-induced gut barrier dysfunction.

Extrathoracic multiple trauma dysregulates neutrophil function and exacerbates pneumonia-induced lung injury.

BACKGROUND: Forty percent of critically ill trauma patients will develop an infectious complication. Pneumonia is the most common cause of death of trauma patients surviving their initial insult. We previously demonstrated that polytrauma (PT), defined as two or more severe injuries in at least two areas of the body, induces emergency hematopoiesis characterized by accelerated myelopoiesis in the bone marrow and increased myeloid cell frequency in the peripheral tissues. We hypothesized that PT alone induces priming of neutrophils, resulting in hyperactivation upon secondary exposure to bacteria and causing acute lung injury and increased susceptibility to secondary exposure to Pseudomonas aeruginosa pneumonia. METHODS: C57BL/6 mice were subjected to PT consisting of a lower extremity pseudofracture, liver crush injury, and 15% blood-volume hemorrhage. Pneumonia was induced by intratracheal injection of 5 × 106 CFU live P. aeruginosa or 1 × 107 of heat-killed P. aeruginosa (HKPA). For reactive oxygen species (ROS), studies polymorphonuclear neutrophils (PMNs) were isolated by immunomagnetic bead negative selection and stimulated ex-vivo with HKPA. Reactive oxygen species production was measured by immunofluorescence. For histology, lung sections were stained by hematoxylin-eosin and analyzed by a blinded grader. RESULTS: Polytrauma induced persistent changes in immune function at baseline and to secondary infection. Pneumonia after injury resulted in increased mortality (60% vs. 5% p < 0.01). Blood neutrophils from PT mice had higher resting (unstimulated) ROS production than in naive animals (p < 0.02) demonstrating priming of the neutrophils following PT. After intratracheal HKPA injection, bronchoalveolar lavage PMNs from injured mice had higher ROS production compared with naive mice (p < 0.01), demonstrating an overexuberant immunopathologic response of neutrophils following PT. CONCLUSION: Polytrauma primes neutrophils and causes immunopathologic PMN ROS production, increased lung injury and susceptibility to secondary bacterial pneumonia. These results suggest that trauma-induced immune dysfunction can cause immunopathologic response to secondary infection and suggests neutrophil-mediated pulmonary damage as a therapeutic target for posttrauma pneumonia.

In Vitro-Administered Dexamethasone Suppresses T Cell Function With Reversal by Interleukin-7 in Coronavirus Disease 2019.

OBJECTIVES: Corticosteroid therapy has become standard of care therapy for hospitalized patients infected with the severe acute respiratory syndrome coronavirus-2 global pandemic-causing virus. Whereas systemic inflammation is a notably important feature in coronavirus disease 2019 pathogenesis, adaptive immune suppression and the inability to eradicate effectively the virus remain significant factors as well. We sought to evaluate the in vitro effects of dexamethasone phosphate on T cell function in peripheral blood mononuclear cells derived from patients with acute, severe, and moderate coronavirus disease 2019. DESIGN: Prospective in vitro laboratory study. SETTING: Coronavirus disease 2019-specific medical wards and ICUs at a single-center, quaternary-care academic hospital between October 1, 2020, and November 15, 2020. PATIENTS: Eleven patients diagnosed with coronavirus disease 2019 admitted to either the ICU or hospital coronavirus disease 2019 unit. Three patients had received at least one dose of dexamethasone prior to enrollment. INTERVENTIONS: Fresh whole blood was collected, and peripheral blood mononuclear cells were immediately isolated and plated onto precoated enzyme-linked immunospot plates for detection of interferon-γ production. Samples were incubated with CD3/CD28 antibodies alone and with three concentrations of dexamethasone. These conditions were also stimulated with recombinant human interleukin-7. Following overnight incubation, the plates were washed and stained for analysis using Cellular Technology Limited ImmunoSpot S6 universal analyzer (ImmunoSpot by Cellular Technology Limited, Cleveland, OH). MEASUREMENTS AND MAIN RESULTS: Functional cytokine production was assessed by quantitation of cell spot number and total well intensity after calculation for each enzyme-linked immunospot well using the Cellular Technology Limited ImmunoSpot Version 7.0 professional software (CTL Analyzers, Shaker Heights, OH). Comparisons were made using t test and using a nonparametric analysis of variance Friedman test. The number of functional T cells producing interferon-γ and the intensity of the response decrease significantly with exposure to 1.2-µg/mL dexamethasone. About 0.12 µg/mL does not significantly affect the functional immune response on enzyme-linked immunospot. Interleukin-7 increases the overall number of activated T cells, including those exposed to dexamethasone. CONCLUSIONS: Further evaluation of the effect of immunomodulatory therapies is warranted in coronavirus disease 2019. A refined functional, precision medicine approach that evaluates the cellular immune function of individual patients with coronavirus disease 2019 is needed to better define which therapies could have benefit or cause harm for specific patients.

Dysregulation of the Leukocyte Signaling Landscape during Acute COVID-19.

The global COVID-19 pandemic has claimed the lives of more than 450,000 US citizens. Dysregulation of the immune system underlies the pathogenesis of COVID-19, with inflammation mediated local tissue injury to the lung in the setting of suppressed systemic immune function. To define the molecular mechanisms of immune dysfunction in COVID-19 we utilized a systems immunology approach centered on the circulating leukocyte phosphoproteome measured by mass cytometry. COVID-19 is associated with wholesale activation of a broad set of signaling pathways across myeloid and lymphoid cell populations. STAT3 phosphorylation predominated in both monocytes and T cells and was tightly correlated with circulating IL-6 levels. High levels of STAT3 phosphorylation was associated with decreased markers of myeloid cell maturation/activation and decreased ex-vivo T cell IFN-gamma production, demonstrating that during COVID-19 dysregulated cellular activation is associated with suppression of immune effector cell function. Collectively, these data reconcile the systemic inflammatory response and functional immunosuppression induced by COVID-19 and suggest STAT3 signaling may be the central pathophysiologic mechanism driving immune dysfunction in COVID-19.

SAVE 2.0: Identifying and strengthening resident leadership skills through simulation based team training.

BACKGROUND: The "Surgery for Abdomino-thoracic ViolencE (SAVE)" animate lab engages surgical residents in the management of penetrating injuries in a team setting. Senior residents, representing postgraduate year (PGY) 3-5, assume the role of team leader and facilitate the junior residents, PGY1-2, in operative management of simulated penetrating wounds. Residents completed five scenarios with increasing level of difficulty within set time limits. Senior residents were evaluated on their team's ability to "SAVE" their patient within the time allotted, as well as their communication and leadership skills. METHODS: General, vascular, urology, and plastic surgery residents (n = 79) were divided into 25 teams of three to four residents by "resident scores" (R scores, the sum of the team members' PGY) to create balanced teams with comparable years of clinical experience. Residents completed assessments of their senior resident's leadership ability and style. RESULTS: Evaluation of a resident's desired learning style changed across PGY with junior residents preferring more hands-on guidance compared with senior residents preferring only verbal correction. Resident leadership evaluations demonstrated that team leaders of varied resident years achieved the highest scores. Greater differences in the mismatch between autonomy provided to and desired by junior residents correlated to greater junior resident discomfort in expressing their opinion, confidence, and leadership ratings of senior residents. However, greater autonomy mismatch also correlated to more rapid time to task completion. CONCLUSION: Different from our expectations, clinical experience alone did not define team leader success. Leadership is a powerful influence on the outcome of team performance and may be a skill, which can transcend overall clinical experience. A match between desired and provided resident autonomy and team cohesion may demonstrate a stronger effect on team success in stressful operative situations, such as trauma resuscitation. Enhancement of leadership skills early in residency training may represent an important focus for trauma surgery education.

A Whole Blood Enzyme-Linked Immunospot Assay for Functional Immune Endotyping of Septic Patients.

Sepsis initiates simultaneous pro- and anti-inflammatory processes, the pattern and intensity of which vary over time. The inability to evaluate the immune status of patients with sepsis in a rapid and quantifiable manner has undoubtedly been a major reason for the failure of many therapeutic trials. Although there has been considerable effort to immunophenotype septic patients, these methods have often not accurately assessed the functional state of host immunity, lack dynamic range, and are more reflective of molecular processes rather than host immunity. In contrast, ELISpot assay measures the number and intensity of cytokine-secreting cells and has excellent dynamic range with rapid turnaround. We investigated the ability of a (to our knowledge) novel whole blood ELISpot assay and compared it with a more traditional ELISpot assay using PBMCs in sepsis. IFN-γ and TNF-α ELISpot assays on whole blood and PBMCs were undertaken in control, critically ill nonseptic, and septic patients. Whole blood ELISpot was easy to perform, and results were generally comparable to PBMC-based ELISpot. However, the whole blood ELISpot assay revealed that nonmonocyte, myeloid populations are a significant source of ex vivo TNF-α production. Septic patients who died had early, profound, and sustained suppression of innate and adaptive immunity. A cohort of septic patients had increased cytokine production compared with controls consistent with either an appropriate or excessive immune response. IL-7 restored ex vivo IFN-γ production in septic patients. The whole blood ELISpot assay offers a significant advance in the ability to immunophenotype patients with sepsis and to guide potential new immunotherapies.

Macrophage-Derived Vascular Endothelial Growth Factor-A Is Integral to Neuromuscular Junction Reinnervation after Nerve Injury.

Functional recovery in the end target muscle is a determinant of outcome after peripheral nerve injury. The neuromuscular junction (NMJ) provides the interface between nerve and muscle and includes non-myelinating terminal Schwann cells (tSCs). After nerve injury, tSCs extend cytoplasmic processes between NMJs to guide axon growth and NMJ reinnervation. The mechanisms related to NMJ reinnervation are not known. We used multiple mouse models to investigate the mechanisms of NMJ reinnervation in both sexes, specifically whether macrophage-derived vascular endothelial growth factor-A (Vegf-A) is crucial to establishing NMJ reinnervation at the end target muscle. Both macrophage number and Vegf-A expression increased in end target muscles after nerve injury and repair. In mice with impaired recruitment of macrophages and monocytes (Ccr2-/- mice), the absence of CD68+ cells (macrophages) in the muscle resulted in diminished muscle function. Using a Vegf-receptor 2 (VegfR2) inhibitor (cabozantinib; CBZ) via oral gavage in wild-type (WT) mice resulted in reduced tSC cytoplasmic process extension and decreased NMJ reinnervation compared with saline controls. Mice with Vegf-A conditionally knocked out in macrophages (Vegf-Afl/fl; LysMCre mice) demonstrated a more prolonged detrimental effect on NMJ reinnervation and worse functional muscle recovery. Together, these results show that contributions of the immune system are integral for NMJ reinnervation and functional muscle recovery after nerve injury.SIGNIFICANCE STATEMENT This work demonstrates beneficial contributions of a macrophage-mediated response for neuromuscular junction (NMJ) reinnervation following nerve injury and repair. Macrophage recruitment occurred at the NMJ, distant from the nerve injury site, to support functional recovery at the muscle. We have shown hindered terminal Schwann cell (tSC) injury response and NMJ recovery with inhibition of: (1) macrophage recruitment after injury; (2) vascular endothelial growth factor receptor 2 (VegfR2) signaling; and (3) Vegf secretion from macrophages. We conclude that macrophage-derived Vegf is a key component of NMJ recovery after injury. Determining the mechanisms active at the end target muscle after motor nerve injury reveals new therapeutic targets that may translate to improve motor recovery following nerve injury.

The Immunologic Effect of Early Intravenous Two and Four Gram Bolus Dosing of Tranexamic Acid Compared to Placebo in Patients With Severe Traumatic Bleeding (TAMPITI): A Randomized, Double-Blind, Placebo-Controlled, Single-Center Trial.

Background: The hemostatic properties of tranexamic acid (TXA) are well described, but the immunological effects of TXA administration after traumatic injury have not been thoroughly examined. We hypothesized TXA would reduce monocyte activation in bleeding trauma patients with severe injury. Methods: This was a single center, double-blinded, randomized controlled trial (RCT) comparing placebo to a 2 g or 4 g intravenous TXA bolus dose in trauma patients with severe injury. Fifty patients were randomized into each study group. The primary outcome was a reduction in monocyte activation as measured by human leukocyte antigen-DR isotype (HLA-DR) expression on monocytes 72 h after TXA administration. Secondary outcomes included kinetic assessment of immune and hemostatic phenotypes within the 72 h window post-TXA administration. Results: The trial occurred between March 2016 and September 2017, when data collection ended. 149 patients were analyzed (placebo, n = 50; 2 g TXA, n = 49; 4 g TXA, n = 50). The fold change in HLA-DR expression on monocytes [reported as median (Q1-Q3)] from pre-TXA to 72 h post-TXA was similar between placebo [0.61 (0.51-0.82)], 2 g TXA [0.57 (0.47-0.75)], and 4 g TXA [0.57 (0.44-0.89)] study groups (p = 0.82). Neutrophil CD62L expression was reduced in the 4 g TXA group [fold change: 0.73 (0.63-0.97)] compared to the placebo group [0.97 (0.78-1.10)] at 24 h post-TXA (p = 0.034). The fold decrease in plasma IL-6 was significantly less in the 4 g TXA group [1.36 (0.87-2.42)] compared to the placebo group [0.46 (0.19-1.69)] at 72 h post-TXA (p = 0.028). There were no differences in frequencies of myeloid or lymphoid populations or in classical complement activation at any of the study time points. Conclusion: In trauma patients with severe injury, 4 g intravenous bolus dosing of TXA has minimal immunomodulatory effects with respect to leukocyte phenotypes and circulating cytokine levels. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT02535949.

Ethics of Codes and Codes of Ethics: When Is It Ethical to Provide Cardiopulmonary Resuscitation During the COVID-19 Pandemic?

OBJECTIVE: Our study aims to provide a paradigm when it is ethical to perform cardiopulmonary resuscitation (CPR) on patients during the COVID-19 pandemic. SUMMARY BACKGROUND DATA: Hospitals around the nation are enacting systems to limit CPR in caring for COVID+ patients for a variety of legitimate reasons and based on concepts of medical futility and allocation of scarce resources. No ethical framework, however, has been proposed as a standard to guide care in this crucial matter. METHODS: Our analysis begins with definitions of ethically relevant terms. We then cycle an illustrative clinical vignette through the mathematically permissible possibilities to account for all conceivable scenarios. Scenarios with ethical tension are examined. RESULTS: Patients have the negative right to refuse care including CPR, but they do not have the positive right to demand it. Our detailed ethical analysis and recommendations support CPR if and only if 1) CPR is judged medically beneficial, and in line with the patient's and values and goals, 2) allocations or scarce resources follow a just and transparent triage system, and 3) providers are protected from contracting the disease. CONCLUSIONS: CPR is an intervention like any other, with attendant risks and benefits and with responsibility for the utilization of limited resources. Our ethical analysis advocates for a systematic approach to codes that respects the important ethical considerations in caring for the critically ill and facilitates patient-centered, evidence-based, and fair treatment to all.