Search

Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ('SPIES') is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients., Competing Interests: Declaration of competing interest M.B.M., K.E.R., and I.R.T. are members of Immune Functional Diagnostics, LLC (IFDx LLC) and receive no direct financial compensation. IFDx LLC is developing predictive metrics in critical illness and this technology is evaluated in this research. S.C·B, L.L.M., R.S.H., and the University of Florida may receive royalty income based on a technology developed by S.C.B. and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research. C.C.C. and the University of Cincinnati may receive royalty income based on a technology developed by C.C.C. and others and licensed by Washington University in St. Louis to IFDx LLC. That technology is evaluated in this research., (Published by Elsevier B.V.)

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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

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., (© 2022. The Author(s), under exclusive licence to Scoliosis Research Society.)

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-1β (IL-1β) 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-1β 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-1β 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-1β expression., (© 2022 by The American Society of Hematology.)

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., Competing Interests: IRT, RSH, KER and MBM have pending intellectual property related to the ELISpot assay. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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., Competing Interests: Decleration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: Dr. Turnbull has received funding from the National Institute of Health (NIH), National Institute of General Medical Sciences (NIGMS) GM133756. Dr. Morre is chief scientist for RevImmune, the company that generously provided interleukin-7 for this patient. Dr. Moldawer has received funding from the NIH, NIGMS GM139046. Dr. Di Paola has received funding from the NIH, National Heart, Lung, Blood Institute HL139825. Dr. Hotchkiss has received funding from the NIH, NIGMS GM126928. Dr. Remy has received funding from the NIH, NIGMS GM129763, and the National Center for Advancing Translational Sciences UL1TR0002345. The remaining authors have disclosed that they do not have any conflicts of interest., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

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., (Copyright © 2021 American Association for the Surgery of Trauma.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

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., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

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.

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., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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.

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., (Copyright © 2020 Spinella, Thomas, Turnbull, Fuchs, Bochicchio, Schuerer, Reese, Coleoglou Centeno, Horn, Baty, Shea, Meledeo, Pusateri, Levy, Cap and Bochicchio.)

COVID-19-associated morbidity and mortality have been attributed to a pathologic host response. Two divergent hypotheses have been proposed: hyperinflammatory cytokine storm; and failure of host protective immunity that results in unrestrained viral dissemination and organ injury. A key explanation for the inability to address this controversy has been the lack of diagnostic tools to evaluate immune function in COVID-19 infections. ELISpot, a highly sensitive, functional immunoassay, was employed in 27 patients with COVID-19, 51 patients with sepsis, 18 critically ill nonseptic (CINS) patients, and 27 healthy control volunteers to evaluate adaptive and innate immune status by quantitating T cell IFN-ɣ and monocyte TFN-α production. Circulating T cell subsets were profoundly reduced in COVID-19 patients. Additionally, stimulated blood mononuclear cells produced less than 40%-50% of the IFN-ɣ and TNF-α observed in septic and CINS patients, consistent with markedly impaired immune effector cell function. Approximately 25% of COVID-19 patients had increased IL-6 levels that were not associated with elevations in other canonical proinflammatory cytokines. Collectively, these findings support the hypothesis that COVID-19 suppresses host functional adaptive and innate immunity. Importantly, IL-7 administered ex vivo restored T cell IFN-ɣ production in COVID-19 patients. Thus, ELISpot may functionally characterize host immunity in COVID-19 and inform prospective therapies.

Long-term survival after lung transplantation remains profoundly limited by graft rejection. Recent work has shown that bronchus-associated lymphoid tissue (BALT), characterized by the development of peripheral nodal addressin (PNAd)-expressing high endothelial venules and enriched in B and Foxp3 + T cells, is important for the maintenance of allograft tolerance. Mechanisms underlying BALT induction in tolerant pulmonary allografts, however, remain poorly understood. Here, we show that the development of PNAd-expressing high endothelial venules within intragraft lymphoid follicles and the recruitment of B cells, but not Foxp3 + cells depends on IL-22. We identify graft-infiltrating gamma-delta (γδ) T cells and Type 3 innate lymphoid cells (ILC3s) as important producers of IL-22. Reconstitution of IL-22 at late time points through retransplantation into wildtype hosts mediates B cell recruitment into lymphoid follicles within the allograft, resulting in a significant increase in their size, but does not induce PNAd expression. Our work has identified cellular and molecular requirements for the induction of BALT in pulmonary allografts during tolerance induction and may provide a platform for the development of new therapies for lung transplant patients., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The inflammatory response to infection or injury dramatically increases the hematopoietic demand on the bone marrow to replace effector leukocytes consumed in the inflammatory response. In the setting of infection, pathogen-associated molecular patterns induce emergency hematopoiesis, activating hematopoietic stem and progenitor cells to proliferate and produce progeny for accelerated myelopoiesis. Sterile tissue injury due to trauma also increases leukocyte demand; however, the effect of sterile tissue injury on hematopoiesis is not well described. We find that tissue injury alone induces emergency hematopoiesis in mice subjected to polytrauma. This process is driven by IL-1/MyD88-dependent production of G-CSF. G-CSF induces the expansion of hematopoietic progenitors, including hematopoietic stem cells and multipotent progenitors, and increases the frequency of myeloid-skewed progenitors. To our knowledge, these data provide the first comprehensive description of injury-induced emergency hematopoiesis and identify an IL-1/MyD88/G-CSF-dependent pathway as the key regulator of emergency hematopoiesis after injury., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Background: Previous studies have suggested that percutaneous drainage and interval appendectomy is an effective treatment for appendicitis with associated abscess. Few studies to date have analyzed risk factors for failed drain management. We hypothesized that older patients with more co-morbidities would be at higher risk for failing conservative treatment., Methods: The 2010-2014 editions of the National Inpatient Sample (NIS) were queried for patients with diagnoses of peri-appendiceal abscesses. Minors and elective admissions were excluded. We identified patients who underwent percutaneous drainage and defined drain failure as undergoing a surgical operation after drainage but during the same inpatient visit to assess for factors associated with failure of drainage alone as a treatment. After univariable analysis, binomial logistic regression was used to assess for independent risk factors. Frequencies were analyzed by χ 2 and continuous variables by Student's t-test., Results: A total of 2,209 patients with appendiceal abscesses received drains; 561 patients (25.4%) failed conservative management and underwent operative intervention. On univariable analysis, patients who failed conservative management were younger, more likely to be Hispanic, have more inpatient diagnoses, and to have undergone drainage earlier in the hospital course. Multivariable regression demonstrated that the number of diagnoses, female sex, and Hispanic race were predictive of failure of drainage alone. Older age, West and Midwest census regions, and later drain placement were predictive of successful treatment with drainage alone. Failure was associated with more charges and longer hospital stay but not with a higher mortality rate., Conclusion: Approximately a quarter of patients will fail management of appendiceal abscess with percutaneous drain placement alone. Risk factors for failure are patient complexity, female sex, earlier drainage, and Hispanic race. Failure of drainage is associated with higher total charges and longer hospital stay; however, no change in the mortality rate was noted.

Background: Appendicitis is the most common intraabdominal surgical emergency in the United States, with over 250,000 cases each year. Several recent studies have evaluated the efficacy of nonoperative management of appendicitis. We measured changes in the treatment of appendicitis in the United States from 1998 to 2014 and evaluated outcomes in the contemporary cohort of appendicitis cases from 2010 to 2014., Methods: The National Inpatient Sample was queried for cases with a principal diagnosis of appendicitis. Cases with peritoneal abscesses were excluded. We determined trends in management and then compared cases managed nonoperatively versus those managed with early operation for demographics and outcomes including mortality, total charges, and length of stay using univariate analysis, binary logistic regression analysis, and case-control matching., Results: Although early operation remains the dominant treatment for acute appendicitis in the United States, there is an accelerating trend in nonoperative management. Nonoperative management is associated with increased age, number of comorbidities, and inpatient diagnoses. In univariate, multiple regression, and case-control analysis, nonoperative management is associated with decreased total charges but significantly increased risk of mortality., Conclusions: Elderly patients and patients with medical comorbidities are more likely to be treated nonoperatively for appendicitis than younger patients. Although previously published data support nonoperative management of appendicitis in low-risk surgical patients, we suggest that elderly or medically complex patients may benefit from early operative treatment of appendicitis and are potentially at risk of poor outcomes from nonoperative management., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. Early recognition and treatment are the cornerstones of management., Methods: Review of the English-language literature., Results: For both sepsis and septic shock "antimicrobials [should be] be initiated as soon as possible and within one hour" (Surviving Sepsis Campaign). The risk of progression from severe sepsis to septic shock increases 8% for each hour before antibiotics are started. Selection of antimicrobial agents is based on a combination of patient factors, predicted infecting organism(s), and local microbial resistance patterns. The initial drugs should have activity against typical gram-positive and gram-negative causative micro-organisms. Anaerobic coverage should be provided for intra-abdominal infections or others where anaerobes are significant pathogens. Empiric antifungal or antiviral therapy may be warranted. For patients with healthcare-associated infections, resistant micro-organisms will further complicate the choice of empiric antimicrobials. Recommendations are given for specific infections., Conclusion: Early administration of broad-spectrum antimicrobial drugs is one of the most important, if not the most important, treatment for patients with sepsis or septic shock. Drugs should be initiated as soon as possible, and the choice of should take into account patient factors, common local pathogens, hospital antibiograms and resistance patterns, and the suspected source of infection. Antimicrobial agent therapy should be de-escalated as soon as possible.

Background: Antibiotic-impregnated central venous catheters (CVCs) decrease the incidence of infection in high-risk patients. However, use of these catheters carries the hypothetical risk of inducing antibiotic resistance. We hypothesized that routine use of minocycline and rifampin-impregnated catheters (MR-CVC) in a single intensive care unit (ICU) would change the resistance profile for Staphylococcus aureus., Methods: We reviewed antibiotic susceptibilities of S. aureus isolates obtained from blood cultures in a large urban teaching hospital from 2002-2015. Resistance patterns were compared before and after implementation of MR-CVC use in the surgical ICU (SICU) in August 2006. We also compared resistance patterns of S. aureus obtained in other ICUs and in non-ICU patients, in whom MR-CVCs were not used., Results: Data for rifampin, oxacillin, and clindamycin were available for 9,703 cultures; tetracycline resistance data were available for 4,627 cultures. After implementation of MR-CVC use in the SICU, rifampin resistance remained unchanged, with rates the same as in other ICU and non-ICU populations (3%). After six years of use of MR-CVCs in the SICU, the rate of tetracycline resistance was unchanged in all facilities (1%-3%). The use of MR-CVCs was not associated with any change in S. aureus oxacillin-resistance rates in the SICU (66% vs. 60%). However, there was a significant decrease in S. aureus clindamycin resistance (59% vs. 34%; p < 0.05) in SICU patients., Conclusions: Routine use of rifampin-minocycline-impregnated CVCs in the SICU was not associated with increased resistance of S. aureus isolates to rifampin or tetracyclines.

Background: Necrotizing soft-tissue infections (NSTIs) result in significant morbidity and mortality rates, with as many as 76% of patients dying during their index admission. Published data suggest NSTIs rarely involve fungal infections in immunocompetent patients. However, because of the recent recognition of fungal infections in our population, we hypothesized that such infections frequently complicate NSTIs and are associated with higher morbidity and mortality rates., Methods: A prospectively maintained Acute and Critical Care Surgery (ACCS) database spanning 2008-2015 and including more than 7,000 patients was queried for patients with NSTIs. Microbiologic data, demographics, and clinical outcomes were abstracted. Risk factors and outcomes associated with NSTI with positive intra-operative fungal cultures were determined. Frequencies were compared by χ 2 and continuous variables by the Student t-test using SPSS. Because the study included only archived data, no patient permission was needed., Results: A total of 230 patients were found to have NSTIs; 197 had intra-operative cultures, and 21 (10.7%) of these were positive for fungi. Fungal infection was more common in women, patients with higher body mass index (BMI), and patients who had had prior abdominal procedures. There were no significant differences in demographics, co-morbidities, or site of infection. The majority of patients (85.7%) had mixed bacterial and fungal infections; in the remaining patients, fungi were the only species isolated. Most fungal cultures were collected within 48 h of hospital admission, suggesting that the infections were not hospital acquired. Patients with positive fungal cultures required two more surgical interventions and had a three-fold greater mortality rate than patients without fungal infections., Conclusions: This is the largest series to date describing the impact of fungal infection in NSTIs. Our data demonstrate a three-fold increase in the mortality rate and the need for two additional operations. Consideration should be given to starting patients on empiric anti-fungal therapy in certain circumstances.

Background: Burnout is a complex syndrome of emotional distress that can disproportionately affect individuals who work in health care professions., Study Design: For a national survey of burnout in US general surgery residents, we asked all ACGME-accredited general surgery program directors to email their general surgery residents an invitation to complete an anonymous, online survey. Burnout was assessed with the Maslach Burnout Inventory; total scores for Emotional Exhaustion (EE), Depersonalization (DP), and Personal Accomplishment (PA) subscales were calculated. Burnout was defined as having a score in the highest tertile for EE or DP or lowest tertile for PA. Chi-square tests and one-way ANOVA were used to test associations between burnout tertiles for each subscale and various resident and training-program characteristics as appropriate., Results: From April to December 2014, six hundred and sixty-five residents actively engaged in clinical training had data for analysis; 69% met the criterion for burnout on at least one subscale. Higher burnout on each subscale was reported by residents planning private practice careers compared with academic careers. A greater proportion of women than men reported burnout on EE and PA. Higher burnout on EE and DP was associated with greater work hours per week. Having a structured mentoring program was associated with lower burnout on each subscale., Conclusions: The high rates of burnout among general surgery residents are concerning, given the potential impact of burnout on the quality of patient care. Efforts to identify at-risk populations and to design targeted interventions to mitigate burnout in surgical trainees are warranted., (Copyright © 2016 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Objectives: Obesity is associated with a hypercoagulable state at baseline and following injury. The anatomic location of adipose deposition may influence the type of thrombotic event, with visceral adipose tissue (VAT) associated with arterial thrombosis and subcutaneous adipose tissue (SAT) predisposing to venous thrombosis. We sought to determine whether adipose tissue amount and location correlated with measures of coagulation., Methods: All adult Level I trauma activations at our institution between January 2013 and August 2014 who underwent admission abdominal computed tomography scan and had admission rotational thromboelastometry measurements were included. Patients were excluded for history of anticoagulant use and known coagulopathy/hypercoagulable state. Admission computed tomography was used to obtain cross-sectional VAT and SAT areas at the umbilicus utilizing a novel software system; VAT and SAT measurements were associated with markers of coagulation utilizing Spearman correlation and stepwise linear regression with significance set at p < 0.05., Results: Two hundred forty-two patients met inclusion and exclusion criteria. Sixty-nine percent of patients sustained blunt injury, 79% were male, mean age was 40 years, 25% were obese or morbidly obese, and mean Injury Severity Scale score was 17. Seventeen percent of patients had acute deep venous thrombosis or pulmonary embolism during hospitalization. Neither SAT nor VAT correlated with prothrombin time, international normalized ratio, or partial thromboplastin time. Subcutaneous adipose tissue correlated positively with platelet count. Visceral adipose tissue and SAT correlated negatively with clot formation time and positively with TEM fibrinogen, α angle, maximum clot firmness, and lysis at 30 minutes; stronger correlations and greater significance were seen between SAT and these measures except for lysis at 30 minutes. Stepwise linear regression confirmed significant relationships between SAT and clot formation time, AA, and maximum clot firmness; VAT showed a significant relationship with TEM fibrinogen., Conclusions: Increased adipose tissue correlates with relative hypercoagulability following trauma. Subcutaneous adipose tissue shows a stronger relationship with functional measures of coagulation, suggesting that SAT may be associated with hemorrhage resistance and hypercoagulability after injury., Level of Evidence: Prognostic study, level IV.

For more than two decades, sepsis was defined as a microbial infection that produces fever (or hypothermia), tachycardia, tachypnoea and blood leukocyte changes. Sepsis is now increasingly being considered a dysregulated systemic inflammatory and immune response to microbial invasion that produces organ injury for which mortality rates are declining to 15-25%. Septic shock remains defined as sepsis with hyperlactataemia and concurrent hypotension requiring vasopressor therapy, with in-hospital mortality rates approaching 30-50%. With earlier recognition and more compliance to best practices, sepsis has become less of an immediate life-threatening disorder and more of a long-term chronic critical illness, often associated with prolonged inflammation, immune suppression, organ injury and lean tissue wasting. Furthermore, patients who survive sepsis have continuing risk of mortality after discharge, as well as long-term cognitive and functional deficits. Earlier recognition and improved implementation of best practices have reduced in-hospital mortality, but results from the use of immunomodulatory agents to date have been disappointing. Similarly, no biomarker can definitely diagnose sepsis or predict its clinical outcome. Because of its complexity, improvements in sepsis outcomes are likely to continue to be slow and incremental.

Pneumonia is the most common complication observed in patients with severe injuries. Although the average age of injured patients is 47 years, existing studies of the effect of injury on the susceptibility to infectious complications have focused on young animals, equivalent to a late adolescent human. We hypothesized that mature adult animals are more susceptible to infection after injury than younger counterparts. To test this hypothesis, we challenged 6 to 8-month-old mature mice to a polytrauma injury followed by Pseudomonas aeruginosa pneumonia and compared them to young (8-10-week-old) animals. We demonstrate that polytrauma injury increases mortality from pneumonia in mature animals (sham-pneumonia 21% vs. polytrauma-pneumonia 62%) but not younger counterparts. After polytrauma, pneumonia in mature mice is associated with higher bacterial burden in lung, increased incidence of bacteremia, and elevated levels of bacteria in the blood, demonstrating that injury decreases the ability to control the infectious challenge. We further find that polytrauma did not induce elevations in circulating cytokine levels (TNF-alpha, IL-6, KC, and IL-10) 24  h after injury. However, mature mice subjected to polytrauma demonstrated an exaggerated circulating inflammatory cytokine response to subsequent Pseudomonas pneumonia. Additionally, whereas prior injury increases LPS-stimulated IL-6 production by peripheral blood leukocytes from young (8-10-week-old) mice, injury does not prime IL-6 production by cell from mature adult mice. We conclude that in mature mice polytrauma results in increased susceptibility to Pseudomonas pneumonia while priming an exaggerated but ineffective inflammatory response.

Background: Obese patients are more prone to post-injury multiple organ failure (MOF). Obesity pathophysiology includes an adipose-tissue-derived, renin-angiotensin-aldosterone system affecting inflammatory responses via leukocyte angiotensin receptors. We hypothesized that obese patients receiving pre-injury angiotensin-converting enzyme inhibitor (ACE) or angiotensin receptor blocker (ARB) therapy would have decreased MOF and differences in immune cell frequencies., Study Design: We analyzed the Inflammation and the Host Response to Injury trauma-related database. Patients receiving pre-injury ACE or ARB were stratified as obese (BMI >30 kg/m(2)) or nonobese (BMI <30 kg/m(2)). Groups were age, sex, and Injury Severity Score matched against patients not receiving this therapy. Primary end points were Marshall Multiple Organ Dysfunction Score, Denver-2 Postinjury MOF Score, leukocyte markers on T cells, and monocytes measured by flow cytometry., Results: We evaluated 1,932 patients. One hundred and ten were receiving pre-injury ACE/ARB; 94 patients had data available to calculate BMI. Obese patients receiving ACE/ARB showed maximum Marshall (5.83 ± 2.87) and Denver-2 (2.45 ± 2.32) scores similar to nonobese patients receiving or not receiving ACE/ARB, and obese patients not receiving ACE/ARB had significantly higher Marshall (6.49 ± 2.57; p = 0.009) and Denver-2 (3.33 ± 2.21; p = 0.006) scores. Leukocyte analysis suggested improved T-cell function and monocyte maturation in obese patients on ACE/ARB., Conclusions: Obese patients receiving preinjury ACE/ARB therapy demonstrate post-injury MOF scores similar to nonobese patients; obese patients not receiving these medications have greater post-injury MOF. Leukocyte analysis demonstrates improved immune regulation. Modulation of the renin-angiotensin-aldosterone system pathway might represent a novel therapeutic target in severely injured obese patients., (Copyright © 2015 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Internal fixation of the ribs has been shown in numerous studies to decrease complications following traumatic rib fractures. Anterior injuries to the chest wall causing cartilaginous fractures, although rare, can cause significant disability and can lead to a variety of complications and, therefore, pose a unique clinical problem. Here, we report the surgical technique used for four patients with internal fixation of injuries to the cartilaginous portions of the chest wall treated at our center. All patients had excellent clinical outcomes and reported improvement in symptoms, with no associated complications. Patients who have injuries to the anterior portions of the chest wall should be considered for internal fixation of the chest wall when the injuries are severe and can lead to clinical disability.

Objective: Pathogens that cause pneumonia may be treated in a targeted fashion by antibiotics, but if this therapy fails, then treatment involves only nonspecific supportive measures, independent of the inciting infection. The purpose of this study was to determine whether host response is similar after disparate infections with similar mortalities., Design: Prospective, randomized controlled study., Setting: Animal laboratory in a university medical center., Interventions: Pneumonia was induced in FVB/N mice by either Streptococcus pneumoniae or two different concentrations of Pseudomonas aeruginosa. Plasma and bronchoalveolar lavage fluid from septic animals was assayed by a microarray immunoassay measuring 18 inflammatory mediators at multiple time points., Measurements and Main Results: The host response was dependent on the causative organism as well as kinetics of mortality, but the pro-inflammatory and anti-inflammatory responses were independent of inoculum concentration or degree of bacteremia. Pneumonia caused by different concentrations of the same bacteria, Pseudomonas aeruginosa, also yielded distinct inflammatory responses; however, inflammatory mediator expression did not directly track the severity of infection. For all infections, the host response was compartmentalized, with markedly different concentrations of inflammatory mediators in the systemic circulation and the lungs. Hierarchical clustering analysis resulted in the identification of five distinct clusters of the host response to bacterial infection. Principal components analysis correlated pulmonary macrophage inflammatory peptide-2 and interleukin-10 with progression of infection, whereas elevated plasma tumor necrosis factor sr2 and macrophage chemotactic peptide-1 were indicative of fulminant disease with >90% mortality within 48 hrs., Conclusions: Septic mice have distinct local and systemic responses to Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia. Targeting specific host inflammatory responses induced by distinct bacterial infections could represent a potential therapeutic approach in the treatment of sepsis.

Macrophage colony-stimulating factor (M-CSF) influences the proliferation and survival of mononuclear phagocytes through the receptor CSF-1R. The adaptor protein DAP12 is critical for the function of mononuclear phagocytes. DAP12-mutant mice and humans have defects in osteoclasts and microglia, as well as brain and bone abnormalities. Here we show DAP12 deficiency impaired the M-CSF-induced proliferation and survival of macrophages in vitro. DAP12-deficient mice had fewer microglia in defined central nervous system areas, and DAP12-deficient progenitors regenerated myeloid cells inefficiently after bone marrow transplantation. Signaling by M-CSF through CSF-1R induced the stabilization and nuclear translocation of beta-catenin, which activated genes involved in the cell cycle. DAP12 was essential for phosphorylation and nuclear accumulation of beta-catenin. Our results provide a mechanistic explanation for the many defects of DAP12-deficient mononuclear phagocytes.

Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag-1(-/-) and wild-type (WT) mice. However, Rag-1(-/-) animals have a 5-fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes in Rag-1(-/-) mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4(+) but not CD8(+), gammadelta, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut-specific overexpression of Bcl-2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut-specific overexpression of Bcl-2 fails to alter survival when the transgene is overexpressed in Rag-1(-/-) mice. Further, adoptively transferring lymphocytes to Rag-1(-/-) mice that simultaneously overexpress gut-specific Bcl-2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4(+) lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.

Objective: Aging is associated with increased inflammation following sepsis. The purpose of this study was to determine whether this represents a fundamental age-based difference in the host response or is secondary to the increased mortality seen in aged hosts., Design: Prospective, randomized controlled study., Setting: Animal laboratory in a university medical center., Subjects: Young (6-12 weeks) and aged (20-24 months) FVB/N mice., Interventions: Mice were subjected to 2 x 25 or 1 x 30 cecal ligation and puncture (CLP)., Measurements and Main Results: Survival was similar in young mice subjected to 2 x 25 CLP and aged mice subjected to 1 x 30 CLP (p = 0.15). Young mice subjected to 1 x 30 CLP had improved survival compared with the other groups (p < 0.05). When injury was held constant but mortality was greater, both systemic and peritoneal levels of tumor necrosis factor-alpha, interleukin (IL)-6, IL-10, and monocyte chemotactic protein-1 were elevated 24 hours after CLP in aged animals compared with young animals (p < 0.05). When mortality was similar but injury severity was different, there were no significant differences in systemic cytokines between aged mice and young mice. In contrast, peritoneal levels of tumor necrosis factor-alpha, IL-6, and IL-10 were higher in aged mice subjected to 1 x 30 CLP than young mice subjected to 2 x 25 CLP despite their similar mortalities (p < 0.05). There were no significant differences in either bacteremia or peritoneal cultures when animals of different ages sustained similar injuries or had different injuries with similar mortalities., Conclusions: Aged mice are more likely to die of sepsis than young mice when subjected to an equivalent insult, and this is associated with increases in both systemic and local inflammation. There is an exaggerated local but not systemic inflammatory response in aged mice compared with young mice when mortality is similar. This suggests that systemic processes that culminate in death may be age independent, but the local inflammatory response may be greater with aging.

When associated with different receptors, the signalling adaptor DAP12 has been shown to both potentiate and attenuate the activation of leukocytes. But how can a protein with a single signalling motif elicit qualitatively different cellular responses? We describe a model of DAP12 function, whereby the quality of the cellular response (activation or inhibition) is modulated by the avidity of the interaction between the DAP12-associated receptor and its ligand. This model extends from previous studies of inhibitory signalling mediated by other adaptors, such as the Fc-receptor gamma-chain and CD3zeta, and provides a potential mechanism for the conflicting phenotypes observed in studies of DAP12-deficient mice.

TREM proteins are a family of cell surface receptors that participate in diverse cell processes, including inflammation, bone homeostasis, neurological development and coagulation. TREM-1, the first to be identified, acts to amplify inflammation. Other TREM proteins regulate the differentiation and function of macrophages, microglia, dendritic cells, osteoclasts and platelets. Here we discuss the state of the field, putative ligands of TREM proteins and the challenges that remain in understanding TREM biology.

The triggering receptor expressed on myeloid cells 2 (TREM-2) delivers intracellular signals through the adaptor DAP12 to regulate myeloid cell function both within and outside the immune system. The role of TREM-2 in immunity has been obscured by the failure to detect expression of the TREM-2 protein in vivo. In this study, we show that TREM-2 is expressed on macrophages infiltrating the tissues from the circulation and that alternative activation with IL-4 can induce TREM-2. TREM-2 expression is abrogated by macrophage maturation with LPS of IFN-gamma. Using TREM-2(-/-) mice, we find that TREM-2 functions to inhibit cytokine production by macrophages in response to the TLR ligands LPS, zymosan, and CpG. Furthermore, we find that TREM-2 completely accounts for the increased cytokine production previously reported by DAP12(-/-) macrophages. Taken together, these data show that TREM-2 is expressed on newly differentiated and alternatively activated macrophages and functions to restrain macrophage activation.

Background: In animal and human autopsy studies of sepsis, CD4+ splenocytes either undergo apoptosis or are polarized to the Th2 effector subtype. In mice, these changes occur within 24 hours of the onset of sepsis. Preventing the loss of CD4+ T cells and the Th2-polarization of CD4+ T cells provides a significant survival advantage in mouse models of sepsis. The molecular mechanism(s) for the phenotypic changes of splenic CD4+ T cells in sepsis are not well understood., Study Design: CD4+ splenocytes were enriched by negative selection from disaggregated spleens of septic and sham-operated mice at 6 and 24 hours after surgery. Phenotypic analysis using cell surface markers (CD25, CD44, CD62L, CD69), cytokine secretion in response to CD3/CD28 coligation, and whole genome microarray gene expression profiles were obtained for these cells., Results: Consistent with previous reports, sepsis induced a progressive decrease in the number of CD4+ splenocytes and a time-dependent alteration in CD4+ T-cell phenotype. At 6 hours, when no differences in cell number or surface marker expression were observed, significant alterations in RNA abundance were measured for 498 probe sets. Ontologic classification of these genes indicated changes in cellular physiology. Pathway analysis indicated that T-cell receptor signaling and mitogen-activated protein kinase signaling were significantly altered by sepsis., Conclusions: These data demonstrated a sepsis-specific transcriptional program that precedes sepsis-induced phenotypic changes in CD4+ splenocytes.

The PA200 proteasome activator is a broadly expressed nuclear protein. Although how PA200 normally functions is not fully understood, it has been suggested to be involved in the repair of DNA double-strand breaks (DSBs). The PA200 gene (Psme4) is composed of 45 coding exons spanning 108 kb on mouse chromosome 11. We generated a PA200 null allele (PA200(Delta)) through Cre-loxP-mediated interchromosomal recombination after targeting loxP sites at either end of the locus. PA200(Delta/Delta) mice are viable and have no obvious developmental abnormalities. Both lymphocyte development and immunoglobulin class switching, which rely on the generation and repair of DNA DSBs, are unperturbed in PA200(Delta/Delta) mice. Additionally, PA200(Delta/Delta) embryonic stem cells do not exhibit increased sensitivity to either ionizing radiation or bleomycin. Thus, PA200 is not essential for the repair of DNA DSBs generated in these settings. Notably, loss of PA200 led to a marked reduction in male, but not female, fertility. This was due to defects in spermatogenesis observed in meiotic spermatocytes and during the maturation of postmeiotic haploid spermatids. Thus, PA200 serves an important nonredundant function during spermatogenesis, suggesting that the efficient generation of male gametes has distinct protein metabolic requirements.

Objectives: The aim of this study was to determine the effects of acute lung injury on the gut epithelium and examine mechanisms underlying changes in crypt proliferation and apoptosis. The relationship between severity and timing of lung injury to intestinal pathology was also examined., Design: Randomized, controlled study., Setting: University research laboratory., Subjects: Genetically inbred mice., Interventions: Following induction of acute lung injury, gut epithelial proliferation and apoptosis were assessed in a) C3H/HeN wild-type and C3H/HeJ mice, which lack functional Toll-like receptor 4 (n = 17); b) C57Bl/6 mice that received monoclonal anti-tumor necrosis factor-alpha or control antibody (n = 22); and c) C57Bl/6 wild-type and transgenic mice that overexpress Bcl-2 in their gut epithelium (n = 21). Intestinal epithelial proliferation and death were also examined in animals with differing degrees of lung inflammation (n = 24) as well as in a time course analysis following a fixed injury (n = 18)., Measurements and Main Results: Acute lung injury caused decreased proliferation and increased apoptosis in crypt epithelial cells in all animals studied. C3H/HeJ mice had higher levels of proliferation than C3H/HeN animals without additional changes in apoptosis. Anti-tumor necrosis factor-alpha antibody had no effect on gut epithelial proliferation or death. Overexpression of Bcl-2 did not change proliferation despite decreasing gut apoptosis. Proliferation and apoptosis were not correlated to severity of lung injury, as gut alterations were lost in mice with more severe acute lung injury. Changes in both gut epithelial proliferation and death were apparent within 12 hrs, but proliferation was decreased 36 hrs following acute lung injury while apoptosis returned to normal., Conclusions: Acute lung injury causes disparate effects on crypt proliferation and apoptosis, which occur, at least in part, through differing mechanisms involving Toll-like receptor 4 and Bcl-2. Severity of lung injury does not correlate with perturbations in proliferation or death in the gut epithelium, and acute lung injury-induced changes in intestinal epithelial proliferation persist longer than those in apoptosis.

Background: Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels., Materials and Methods: Hfe-/- mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24-26 months) or mature (16-18 months) Hfe-/- mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels., Results: Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe-/- mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe-/- mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe-/- mice than septic mature Hfe-/- animals. Interleukin-6 was elevated in septic aged Hfe-/- mice compared to sham mice., Conclusions: Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe-/- mice are able to mount an inflammatory response following CLP and mature Hfe-/- mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

DAP12 (KARAP) is a transmembrane signaling adaptor for a family of innate immunoreceptors that have been shown to activate granulocytes and monocytes/macrophages, amplifying production of inflammatory cytokines. Contrasting with these data, recent studies suggest that DAP12 signaling has an inhibitory role in the macrophage response to microbial products (Hamerman, J.A., N.K. Tchao, C.A. Lowell, and L.L. Lanier. 2005. Nat. Immunol. 6:579-586). To determine the in vivo role for DAP12 signaling in inflammation, we measured the response of wild-type (WT) and DAP12-/- mice to septic shock. We show that DAP12-/- mice have improved survival from both endotoxemia and cecal ligation and puncture-induced septic shock. As compared with WT mice, DAP12-/- mice have decreased plasma cytokine levels and a decreased acute phase response during sepsis, but no defect in the recruitment of cells or bacterial control. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augments lipopolysaccharide-mediated cytokine production. These data demonstrate that, during sepsis, DAP12 signaling augments the response to microbial products, amplifying inflammation and contributing to mortality.

Both aging and sepsis independently increase splenic and gut epithelial apoptosis. Sepsis-induced apoptosis in either cell type is also associated with increased mortality in young mice. We sought to determine whether age alters sepsis-induced splenic and gut epithelial cell death. Young (2 months) and aged (22 months) male ND4 mice were subjected to either single-puncture cecal ligation and puncture (CLP) with a 23-gauge needle or sham laparotomy. Apoptosis was assessed 24 hours later in the spleen and gut epithelium by active caspase 3 and hematoxylin and eosin staining. Aged septic mice had increased splenic apoptosis compared with either young septic animals or aged sham animals (15 vs. 7 vs. 5 apoptotic cells/high-powered field, P < 0.05). Similarly, aged septic animals had an elevation in gut epithelial cell death compared with either young septic or aged sham mice (33 vs. 16 vs. 6 apoptotic cells/100 contiguous crypts, P < 0.05). Elevated intestinal cell death was not associated with changes in either gut proliferation or cell division. To verify that the increase in splenic apoptosis seen in septic aged animals was not strain specific, double-puncture CLP with a 25-gauge needle or sham laparotomy was performed on young (4 months) or aged (24 months) C57BL/6 male mice. Similar to results seen in outbred animals, aged septic animals in this inbred strain had increased splenic apoptosis compared with either young septic animals or aged sham animals (23 vs. 7 vs. 4 apoptotic cells/ high powered field, P < 0.05). These results indicate that although infection and aging each independently cause an increase in splenic and gut epithelial apoptosis, their combination leads to a disproportionate increase in cell death in these rapidly dividing cell populations,and potentially plays a role in the marked increase in mortality seen with aging in sepsis.

Objectives: Despite having dysregulated iron metabolism, critically ill patients may receive exogenous iron for the treatment of anemia. Iron is associated with increased tissue apoptosis and may facilitate bacterial growth. We hypothesized that exogenous iron administration given after the onset of sepsis would lead to increased mortality rate. To discriminate between elevated cell death and bacterial overgrowth as potential mediators of mortality, we examined gut epithelial and lymphocyte apoptosis and systemic bacterial counts in animals given iron supplementation after the onset of sepsis., Design: Prospective, randomized, controlled study., Setting: Animal laboratory in a university medical center., Subjects: Male C57BL/6 mice, 6-10 wks old., Interventions: C57BL/6 mice were subjected to cecal ligation and puncture (CLP), a well-accepted model of intra-abdominal sepsis, followed by daily subcutaneous injections of either 1 mL of iron dextran (5 mg/mL) or 0.9% NaCl for a total of five doses. Animals (n = 78) were followed for survival for 8 days. Separate cohorts (n = 76) were killed 24 or 48 hrs after cecal ligation and puncture or sham laparotomy and were assayed for gut epithelial and splenic apoptosis as well as for quantitative blood cultures., Measurements and Main Results: Eight-day survival was 7% in animals that received iron and 26% in mice that received 0.9% NaCl (p < .005). Iron supplementation after cecal ligation and puncture increased apoptosis by both active caspase 3 and hematoxylin and eosin staining in both the intestinal epithelium and spleen at 24 hrs (p < .05). Iron supplementation after sham laparotomy did not cause mortality or elevated apoptosis. Quantitative blood cultures revealed no detectable differences between septic animals that received iron and those that received 0.9% NaCl., Conclusions: High-dose iron supplementation with iron dextran after the onset of sepsis significantly increases mortality rate in this animal model. Iron-induced mortality may be mediated by an increase in gut epithelial and splenic apoptosis, whereas severity of bacteremia does not appear to play a causative role.