TEMPORAL CHANGES IN INNATE AND ADAPTIVE IMMUNITY DURING SEPSIS AS DETERMINED BY ELISPOT.

ABSTRACT: 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 enzyme-linked immunospot (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. Blood and spleens were harvested serially, and ex vivo interferon γ and TNF-α production were compared by ELISpot and enzyme-linked immunosorbent assay. 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 enzyme-linked immunosorbent assay 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.

Whole blood storage duration alters fibrinogen levels and thrombin formation.

INTRODUCTION: Whole blood resuscitation for hemorrhagic shock in trauma represents an opportunity to correct coagulopathy in trauma while also supplying red blood cells. The production of microvesicles in stored whole blood and their effect on its hemostatic parameters have not been described in previous literature. We hypothesized that microvesicles in aged stored whole blood are procoagulant and increase thrombin production via phosphatidylserine. METHODS: Whole blood was obtained from male C57BL/6 male mice and stored in anticoagulant solution for up to 10 days. At intervals, stored whole blood underwent examination with rotational thromboelastography, and platelet-poor plasma was prepared for analysis of thrombin generation. Microvesicles were prepared from 10-day-old whole blood aliquots and added to fresh whole blood or platelet-poor plasma to assess changes in coagulation and thrombin generation. Microvesicles were treated with recombinant mouse lactadherin prior to addition to plasma to inhibit phosphatidylserine's role in thrombin generation. RESULTS: Aged murine whole blood had decreased fibrin clot formation compared with fresh samples with decreased plasma fibrinogen levels. Thrombin generation in plasma from aged blood increased over time of storage. The addition of microvesicles to fresh plasma resulted in increased thrombin generation compared with controls. When phosphatidylserine on microvesicles was blocked with lactadherin, there was no difference in the endogenous thrombin potential, but the generation of thrombin was blunted with lower peak thrombin levels. CONCLUSION: Cold storage of murine whole blood results in decreased fibrinogen levels and fibrin clot formation. Aged whole blood demonstrates increased thrombin generation, and this is due in part to microvesicle production in stored whole blood. One mechanism by which microvesicles are procoagulant is by phosphatidylserine expression on their membranes.

Microvesicles from stored red blood cells induce P-selectin and von Willebrand factor release from endothelial cells via a protein kinase C-dependent mechanism.

INTRODUCTION: The use of packed red blood cells (pRBCs) for resuscitation is limited by the red blood cell storage lesion, a series of biochemical and physiological changes that occur during the storage and aging of blood. Microvesicles (MVs) shed from pRBCs during this process are one component of the red blood cell storage lesion and lead to acute lung injury and pulmonary vascular microthrombi. We hypothesized that MVs from stored pRBCs lead to the release of P-selectin and von Willebrand factor (vWF) from endothelial cells and that this mechanism is mediated via activation of protein kinase C (PKC) or protein kinase A (PKA). METHODS: Leukoreduced, platelet-poor murine pRBCs were isolated from C57BL/6 8-12 week-old male mice via cardiac puncture, prepared via centrifugation using a Ficoll gradient, and stored for up to 14 days, the equivalent of 42 days of storage in humans. MVs were isolated from the stored pRBC units via sequential high-speed centrifugation. Murine lung endothelial cells (MLECs) were cultured and grown to confluence, then treated with MVs and either calphostin C, a PKC inhibitor (10 µg/mL), or PKI 14-22 amide, a PKA inhibitor (10 µM). The supernatant was collected after 1 h. P-selectin and vWF A2 concentrations were quantified via ELISA. Immunofluorescent staining for vWF was performed on MLECs. Statistical analysis was performed via unpaired t-test or ANOVA as indicated and reported as mean ± SD. Concentration is reported as pg/mL. RESULTS: MLECs treated with MVs isolated from stored pRBCs demonstrated increased release of P-selectin and vWF A2 in a dose-dependent fashion. MLECs treated with MVs prepared from stored as compared to fresh pRBCs demonstrated increased release of P-selectin (3751 ± 726 vs 359 ± 64 pg/mL, p < 0.0001) and vWF A2 (3141 ± 355 vs 977 ± 75 pg/mL, p < 0.0001) with increasing duration of storage. The treatment of MVs with calphostin C decreased the amount of P-selectin (1471 ± 444 vs 3751 ± 726 pg/mL, p < 0.0001) and VWF A2 (2401 ± 289 vs 3141 ± 355 pg/mL, p = 0.0017) released into the supernatant by MLECs compared to MVs alone. The treatment of MVs with PKI 14-22 increased the amount of P-selectin released compared to MVs alone (1999 ± 67 vs 1601 ± 135 pg/mL, p = 0.0018). CONCLUSIONS: MVs from stored pRBCs stimulate the release of P-selectin and VWF A2 from endothelial cells. The effect of MVs increases with both dose of MVs and age of stored pRBCs from which they are formed. This mechanism is dependent on activation of PKC and inhibition of this enzyme represents a potentially significant strategy to modulate the inflammatory response to resuscitation with stored pRBCs.

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.

A Prospective Analysis of the SVS WIfI Classification System to Stratify Immediate and 1-Year Patient Outcomes.

The purpose of this study was to prospectively enroll patients that presented to the emergency department with a lower extremity infection, stratify risk and record outcomes. Risk stratification was performed based on the Society of Vascular Surgery Wound, foot Infection, and Ischemia (WIfI) classification system. This study aimed to establish the efficacy and validity of this classification in predicting patient outcomes during immediate hospitalization and throughout a 1 year follow up. A total of 152 patients were enrolled in the study and of these, 116 met the inclusion criteria and had at least 1 year of follow up for analysis. Each patient was assigned a WIfI score based on wound, ischemia, and foot infection severity according to the classification guidelines. Patient demographics as well as all podiatric and vascular procedures were recorded. The major end points of the study were rates of proximal amputation, time to wound healing, surgical procedures, surgical dehiscence, readmission rates, and mortality. A difference in rates of healing (p = .04), surgical dehiscence (p < .01), and 1 year mortality (p = .01) with increasing WIfI stage as well as across the individual component scores was noted. This analysis further supports the application of the WIfI classification system early during patient care to stratify risk and identify the need for early intervention and a multispecialty team approach to potentially improve outcomes in the severe multicomorbid patient.

Heat-killed probiotic Lactobacillus plantarum affects the function of neutrophils but does not improve survival in murine burn injury.

Probiotics have become of interest as therapeutics in trauma or sepsis-induced inflammation due to their ability to affects the immune response. However, their use is still under debate due to the potential risk of septicemia. Therefore, heat-killed probiotics offer a potential alternative, with recent research suggesting a comparable immunomodulating potential and increased safety. In a previous study, we demonstrated decreased mortality by administration of live Lactobacillus plantarum in a mouse burn-sepsis model. Neutrophils are an essential innate defense against pathogens. Therefore, our present study aims to understand the impact of heat-killed probiotic L. plantarum (HKLP) on neutrophil function. Utilizing an in vitro stimulation with HKLP and a burn-infection in vivo model, we determined that administration of HKLP induced significant release of granulocyte-colony stimulating factor (G-CSF) and stimulated the release of pro-and anti-inflammatory cytokines. HKLP had no impact on neutrophil function, such as phagocytosis, oxidative burst, and NETosis, but increased apoptosis and activated neutrophils. HKLP did not improve survival. Together, contrary to our hypothesis, heat-killed probiotics did not improve neutrophil function and survival outcome in a murine severe burn injury model.

Storage with ethanol attenuates the red blood cell storage lesion.

BACKGROUND: Current management of hemorrhagic shock relies on control of surgical bleeding along with resuscitation with packed red blood cells and plasma in a 1-to-1 ratio. Transfusion, however, is not without consequence as red blood cells develop a series of biochemical and physical changes during storage termed "the red blood cell storage lesion." Previous data has suggested that ethanol may stabilize the red blood cell membrane, resulting in improved deformability. We hypothesized that storage of packed red blood cells with ethanol would alter the red blood cell storage lesion. METHODS: Mice underwent donation and storage of red blood cells with standard storage conditions in AS-3 alone or ethanol at concentrations of 0.07%, 0.14%, and 0.28%. The red blood cell storage lesion parameters of microvesicles, Band-3, free hemoglobin, annexin V, and erythrocyte osmotic fragility were measured and compared. In additional experiments, the mice underwent hemorrhage and resuscitation with stored packed red blood cells to further evaluate the in vivo inflammatory impact. RESULTS: Red blood cells stored with ethanol demonstrated decreased microvesicle accumulation and Band-3 levels. There were no differences in phosphatidylserine or cell-free hemoglobin levels. After hemorrhage and resuscitation with packed red blood cells stored with 0.07% ethanol, mice demonstrated decreased serum levels of interleukin-6, macrophage inflammatory protein-1α, keratinocyte chemokine, and tumor necrosis factor α compared to those mice receiving packed red blood cells stored with additive solution-3. CONCLUSION: Storage of murine red blood cells with low-dose ethanol results in decreased red blood cell storage lesion severity. Resuscitation with packed red blood cells stored with 0.07% ethanol also resulted in a decreased systemic inflammatory response in a murine model of hemorrhage.

A digital assay for programmed death-ligand 1 (22C3) quantification combined with immune cell recognition algorithms in non-small cell lung cancer.

PD-L1 (22C3) checkpoint inhibitor therapy represents a mainstay of modern cancer immunotherapy for non-small cell lung cancer (NSCLC). In vitro diagnostic (IVD) PD-L1 antibody staining is widely used to predict clinical intervention efficacy. However, pathologist interpretation of this assay is cumbersome and variable, resulting in poor positive predictive value concerning patient therapy response. To address this, we developed a digital assay (DA) termed Tissue Insight (TI) 22C3 NSCLC, for the quantification of PD-L1 in NSCLC tissues, including digital recognition of macrophages and lymphocytes. We completed clinical validation of this digital image analysis solution in 66 NSCLC patient samples, followed by concordance studies (comparison of PD-L1 manual and digital scores) in an additional 99 patient samples. We then combined this DA with three distinct immune cell recognition algorithms for detecting tissue macrophages, alveolar macrophages, and lymphocytes to aid in sample interpretation. Our PD-L1 (22C3) DA was successfully validated and had a scoring agreement (digital to manual) higher than the inter-pathologist scoring. Furthermore, the number of algorithm-identified immune cells showed significant correlation when compared with those identified by immunohistochemistry in serial sections stained by double immunofluorescence. Here, we demonstrated that TI 22C3 NSCLC DA yields comparable results to pathologist interpretation while eliminating the intra- and inter-pathologist variability associated with manual scoring while providing characterization of the immune microenvironment, which can aid in clinical treatment decisions.

sEH-derived metabolites of linoleic acid drive pathologic inflammation while impairing key innate immune cell function in burn injury.

Fatty acid composition in the Western diet has shifted from saturated to polyunsaturated fatty acids (PUFAs), and specifically to linoleic acid (LA, 18:2), which has gradually increased in the diet over the past 50 y to become the most abundant dietary fatty acid in human adipose tissue. PUFA-derived oxylipins regulate a variety of biological functions. The cytochrome P450 (CYP450)­formed epoxy fatty acid metabolites of LA (EpOMEs) are hydrolyzed by the soluble epoxide hydrolase enzyme (sEH) to dihydroxyoctadecenoic acids (DiHOMEs). DiHOMEs are considered cardioprotective at low concentrations but at higher levels have been implicated as vascular permeability and cytotoxic agents and are associated with acute respiratory distress syndrome in severe COVID-19 patients. High EpOME levels have also correlated with sepsis-related fatalities; however, those studies failed to monitor DiHOME levels. Considering the overlap of burn pathophysiology with these pathologies, the role of DiHOMEs in the immune response to burn injury was investigated. 12,13-DiHOME was found to facilitate the maturation and activation of stimulated neutrophils, while impeding monocyte and macrophage functionality and cytokine generation. In addition, DiHOME serum concentrations were significantly elevated in burn-injured mice and these increases were ablated by administration of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a sEH inhibitor. TPPU also reduced necrosis of innate and adaptive immune cells in burned mice, in a dose-dependent manner. The findings suggest DiHOMEs are a key driver of immune cell dysfunction in severe burn injury through hyperinflammatory neutrophilic and impaired monocytic actions, and inhibition of sEH might be a promising therapeutic strategy to mitigate deleterious outcomes in burn patients.

Functional Characterization of Neutrophils Allows Source Control Evaluation in a Murine Sepsis Model.

INTRODUCTION: Current surgical guidelines for the treatment of intra-abdominal sepsis recommend interventional source control as the key element of therapy, alongside resuscitation and antibiotic administration. Past trials attempted to predict the success of interventional source control to assess whether further interventional therapy is needed. However, no predictive score could be developed. MATERIALS AND METHODS: We utilized an established murine abdominal sepsis model, the cecal ligation and puncture (CLP), and performed a successful surgical source control intervention after full development of sepsis, the CLP-excision (CLP/E). We then sought to evaluate the success of the source control by characterizing circulating neutrophil phenotype and functionality 24 h postintervention. RESULTS: We showed a significant relative increase of neutrophils and a significant absolute and relative increase of activated neutrophils in septic mice. Source control with CLP/E restored these numbers back to baseline. Moreover, main neutrophil functions, the acidification of cell compartments, such as lysosomes, and the production of Tumor Necrosis Factor-alpha (TNF-α), were impaired in septic mice but restored after CLP/E intervention. CONCLUSIONS: Neutrophil characterization by phenotyping and evaluating their functionality indicates successful source control in septic mice and can serve as a prognostic tool. These findings provide a rationale for the phenotypic and functional characterization of neutrophils in human patients with infection. Further studies will be needed to determine whether a predictive score for the assessment of successful surgical source control can be established.

Loss of STING expression is prognostic in non-small cell lung cancer.

BACKGROUND: Stimulator of interferon (IFN) genes (STING) is a protein that promotes type I IFN production essential for T-cell activation. In this study, we aim to characterize STING expression comprehensively using The Cancer Genome Atlas (TCGA) database, cell lines, and patient tumor samples stained with immunohistochemistry. METHODS: Two cohorts were evaluated comprising 721 non-small cell lung cancer (NSCLC) patients and 55 NSCLC cell lines for STING and cyclic GMP-AMP synthase (cGAS) expression using immunohistochemistry. Moreover, an independent cohort of n = 499 patients from the TCGA database was analyzed. Methylation was evaluated on STING and cGAS in five STING-negative NSCLC cell lines. RESULTS: STING RNA expression positively correlates with T cell function and development genes, negatively correlates with cell proliferation and associated with increased survival (5-year-overall survival [OS] 47.3% vs. 38.8%, p = 0.033). STING protein expression is significantly higher in adenocarcinoma (AC) and is lost with increasing stages of AC. STING-positivity is significantly higher in mutant EGFR and KRAS tumors. STING-positive NSCLC patients identified with immunohistochemistry (H-score > 50) have increased survival (median OS: 58 vs. 35 months, p = 0.02). Treatment of STING-negative cell lines with a demethylating agent restores STING expression. CONCLUSIONS: STING is ubiquitously expressed in NSCLC and associated with T cell function genes, AC histology, EGFR, and KRAS mutations and improved overall survival.

Improving packed red blood cell storage with a high-viscosity buffered storage solution.

BACKGROUND: Massive transfusion with older packed red blood cells is associated with increased morbidity and mortality. As packed red blood cells age, they undergo biochemical and structural changes known as the storage lesion. We developed a novel solution to increase viscosity in stored packed red blood cells. We hypothesized that packed red blood cell storage in this solution would blunt storage lesion formation and mitigate the inflammatory response after resuscitation. METHODS: Blood was obtained from 8- to 10-week-old C57BL/6 male donor mice or human volunteers and stored as packed red blood cell units for 14 days for mice or 42 days for humans in either standard AS-3 storage solution or EAS-1587, the novel packed red blood cell storage solution. Packed red blood cells were analyzed for microvesicles, cell-free hemoglobin, phosphatidylserine, band-3 protein, glucose utilization, and osmotic fragility. Additional mice underwent hemorrhage and resuscitation with packed red blood cells stored in either AS-3 or EAS-1587. Serum was analyzed for inflammatory markers. RESULTS: Murine packed red blood cells stored in EAS-1587 demonstrated reductions in microvesicle and cell-free hemoglobin accumulation as well as preserved band-3 expression, increase glucose utilization, reductions in phosphatidylserine expression, and susceptibility to osmotic stress. Serum from mice resuscitated with packed red blood cells stored in EAS-1587 demonstrated reduced proinflammatory cytokines. Human packed red blood cells demonstrated a reduction in microvesicle and cell-free hemoglobin as well as an increase in glucose utilization. CONCLUSION: Storage of packed red blood cells in a novel storage solution mitigated many aspects of the red blood cell storage lesion as well as the inflammatory response to resuscitation after hemorrhage. This modified storage solution may lead to improvement of packed red blood cell storage and reduce harm after massive transfusion.

Survival analysis by inflammatory biomarkers in severely injured patients undergoing damage control resuscitation.

BACKGROUND: Although early balanced blood product resuscitation has improved mortality after traumatic injury, many patients still suffer from inflammatory complications. The goal of this study was to identify inflammatory mediators associated with death and multiorgan system failure following severe injury after patients undergo blood product resuscitation. METHODS: A retrospective secondary analysis of inflammatory markers from the Pragmatic Randomized Optimal Platelet and Plasma Ratios study was performed. Twenty-seven serum biomarkers were measured at 8 time points in the first 72 hours of care and were compared between survivors and nonsurvivors. Biomarkers with significant differences were further analyzed by adjudicated cause of 30-day mortality. RESULTS: Biomarkers from 680 patients were analyzed. Seven key inflammatory markers (IL-1ra, IL-6, IL-8, IL-10, eotaxin, IP-10, and MCP-1) were further analyzed. These cytokines were also noted to have the highest hazard ratios of death. Stepwise selection was used for multivariate analysis of survival by time point. MCP-1 at 2 hours, eotaxin and IP-10 at 12 hours, eotaxin at 24 hours, and IP-10 at 72 hours were associated with all-cause mortality. CONCLUSION: Early systemic inflammatory markers are associated with increased risk of mortality after traumatic injury. Future studies should use these biomarkers to prospectively calculate risks of morbidity and causes of mortality for all trauma patients.

Mild hypobaric hypoxia influences splenic proliferation during the later phase of stress erythropoiesis.

Tissue trauma and hemorrhagic shock are common battlefield injuries that can induce hypoxia, inflammation, and/or anemia. Inflammation and hypoxia can initiate adaptive mechanisms, such as stress erythropoiesis in the spleen, to produce red blood cells and restore the oxygen supply. In a military context, mild hypobaric hypoxia-part of the environmental milieu during aeromedical evacuation or en route care-may influence adaptive mechanisms, such as stress erythropoiesis, and host defense. In the present study, healthy (control), muscle trauma, and polytrauma (muscle trauma and hemorrhagic shock) mice were exposed to normobaric normoxia or hypobaric hypoxia for ∼17.5 h to test the hypothesis that hypobaric hypoxia exposure influences splenic erythropoiesis and splenic inflammation after polytrauma. This hypothesis was partially supported. The polytrauma + hypobaric hypoxia group exhibited more splenic neutrophils, fewer total spleen cells, and fewer splenic proliferating cells than the polytrauma+normobaric normoxia group; however, no splenic erythroid cell differences were detected between the two polytrauma groups. We also compared splenic erythropoiesis and myeloid cell numbers among control, muscle trauma, and polytrauma groups. More reticulocytes at 1.7 days (40 h) post-trauma (dpt) and neutrophils at 4 dpt were produced in the muscle trauma mice than corresponding control mice. In contrast to muscle trauma, polytrauma led to a reduced red blood cell count and elevated serum erythropoietin levels at 1.7 dpt. There were more erythroid subsets and apoptotic reticulocytes in the polytrauma mice than muscle trauma mice at 4 and 8 dpt. At 14 dpt, the red blood cell count of the polytrauma + normobaric normoxia mice was 12% lower than that of the control + normobaric normoxia mice; however, no difference was observed between polytrauma + hypobaric hypoxia and control + hypobaric hypoxia mice. Our findings suggest muscle trauma alone induces stress erythropoiesis; in a polytrauma model, hypobaric hypoxia exposure may result in the dysregulation of splenic cells, requiring a treatment plan to ensure adequate immune functioning.

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.

TPPU treatment of burned mice dampens inflammation and generation of bioactive DHET which impairs neutrophil function.

Oxylipins modulate the behavior of immune cells in inflammation. Soluble epoxide hydrolase (sEH) converts anti-inflammatory epoxyeicosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (DHET). An sEH-inhibitor, TPPU, has been demonstrated to ameliorate lipopolysaccharide (LPS)- and sepsis-induced inflammation via EETs. The immunomodulatory role of DHET is not well characterized. We hypothesized that TPPU dampens inflammation and that sEH-derived DHET alters neutrophil functionality in burn induced inflammation. Outbred mice were treated with vehicle, TPPU or 14,15-DHET and immediately subjected to either sham or dorsal scald 28% total body surface area burn injury. After 6 and 24 h, interleukin 6 (IL-6) serum levels and neutrophil activation were analyzed. For in vitro analyses, bone marrow derived neutrophil functionality and mRNA expression were examined. In vivo, 14,15-DHET and IL-6 serum concentrations were decreased after burn injury with TPPU administration. In vitro, 14,15-DHET impaired neutrophil chemotaxis, acidification, CXCR1/CXCR2 expression and reactive oxygen species (ROS) production, the latter independent from p38MAPK and PI3K signaling. We conclude that TPPU administration decreases DHET post-burn. Furthermore, DHET downregulates key neutrophil immune functions and mRNA expression. Altogether, these data reveal that TPPU not only increases anti-inflammatory and inflammation resolving EET levels, but also prevents potential impairment of neutrophils by DHET in trauma.

Validation of a DKK1 RNAscope chromogenic in situ hybridization assay for gastric and gastroesophageal junction adenocarcinoma tumors.

Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with poor clinical outcomes. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and has demonstrated clinical activity in gastric/gastroesophageal junction (G/GEJ) patients with elevated tumoral expression of DKK1. Here we report on the validation of a DKK1 RNAscope chromogenic in situ hybridization assay to assess DKK1 expression in G/GEJ tumor tissue. To reduce pathologist time, potential pathologist variability from manual scoring and support pathologist decision making, a digital image analysis algorithm that identifies tumor cells and quantifies the DKK1 signal was developed. Following CLIA guidelines the DKK1 RNAscope chromogenic in situ hybridization assay and digital image analysis algorithm were successfully validated for sensitivity, specificity, accuracy, and precision. The DKK1 RNAscope assay in conjunction with the digital image analysis solution is acceptable for prospective screening of G/GEJ adenocarcinoma patients. The work described here will further advance the companion diagnostic development of our DKK1 RNAscope assay and could generally be used as a guide for the validation of RNAscope assays with digital image quantification.

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.

Potential Targets to Mitigate Trauma- or Sepsis-Induced Immune Suppression.

In sepsis and trauma, pathogens and injured tissue provoke a systemic inflammatory reaction which can lead to overwhelming inflammation. Concurrent with the innate hyperinflammatory response is adaptive immune suppression that can become chronic. A current key issue today is that patients who undergo intensive medical care after sepsis or trauma have a high mortality rate after being discharged. This high mortality is thought to be associated with persistent immunosuppression. Knowledge about the pathophysiology leading to this state remains fragmented. Immunosuppressive cytokines play an essential role in mediating and upholding immunosuppression in these patients. Specifically, the cytokines Interleukin-10 (IL-10), Transforming Growth Factor-ß (TGF-ß) and Thymic stromal lymphopoietin (TSLP) are reported to have potent immunosuppressive capacities. Here, we review their ability to suppress inflammation, their dynamics in sepsis and trauma and what drives the pathologic release of these cytokines. They do exert paradoxical effects under certain conditions, which makes it necessary to evaluate their functions in the context of dynamic changes post-sepsis and trauma. Several drugs modulating their functions are currently in clinical trials in the treatment of other pathologies. We provide an overview of the current literature on the effects of IL-10, TGF-ß and TSLP in sepsis and trauma and suggest therapeutic approaches for their modulation.