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BACKGROUND Inflammatory lipid mediators in mesenteric lymph (ML), including arachidonic acid (AA), are considered to play an important role in the pathogenesis of multiple-organ dysfunction after hemorrhagic shock. A previous study suggested that vagus nerve stimulation (VNS) could relieve shock-induced gut injury and abrogate ML toxicity, resulting in the prevention of multiple-organ dysfunction. However, the detailed mechanism of VNS in lymph toxicity remains unclear. The study aimed to investigate the relationship between VNS and inflammatory lipid mediators in ML. METHODS Male Sprague-Dawley rats underwent laparotomy and superior mesenteric artery obstruction (SMAO) for 60 minutes to induce intestinal ischemia followed by reperfusion and observation. The ML duct was cannulated, and ML samples were obtained both before and after SMAO. The distal ileum was removed at the end of the observation period. In one group of animals, VNS was performed from 10 minutes before 10 minutes after SMAO (5 V, 0.5 Hz). Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of AA was performed for each ML sample. The biological activity of ML was examined using a monocyte nuclear factor κ-light-chain-enhancer of activated B cells activation assay. Western blotting of phospholipase A2 group IIA (PLA2-IIA) was also performed for ML and ileum samples. RESULTS Vagus nerve stimulation relieved the SMAO-induced histological gut injury. The concentration of AA and level of nuclear factor κ-light-chain-enhancer of activated B cells activation in ML increased significantly after SMAO, whereas VNS prevented these responses. Western blotting showed PLA2-IIA expression in the ML and ileum after SMAO; however, the appearance of PLA2-IIA band was remarkably decreased in the samples from VNS-treated animals. CONCLUSION The results suggested that VNS could relieve gut injury induced by SMAO and decrease the production of AA in ML by altering PLA2-IIA expression in the gut and ML.

Background Intestinal ischemia caused by hemorrhagic shock is known to induce systemic inflammatory responses. Previous studies have shown that mesenteric lymph (ML) plays a crucial role in gut-mediated inflammation. Lipid mediators, such as lysophosphatidylcholines (LPCs) which contain polyunsaturated fatty acids (PUFAs), are present in the post-shock ML. Exosomes are also present in the ML and act as transcellular carriers of lipids; however, their role in post-shock systemic inflammation has not been revealed. Here, we aimed to identify changes in lipid mediators in ML exosomes after intestinal ischemia. Methods Male Sprague-Dawley rats underwent laparotomy, followed by ML duct cannulation. Animals were subjected to 60 min of intestinal ischemia by superior mesenteric artery clamping, followed by 120 min of reperfusion. ML was obtained before and after intestinal ischemia and exosomes were isolated from ML by ultracentrifugation. The biological activity of ML exosomes was determined using the monocyte NF-κB activation assay. Lipids of ML exosomes were extracted and quantified by liquid chromatography/electrospray ionization mass spectrometry. Results ML exosomes-induced NF-κB activation significantly increased after intestinal ischemia and lipid analysis revealed a significant increase in the concentration of PUFA-containing LPCs. Additionally, PUFA-containing LPCs also induced NF-κB activation. Conclusion Our results suggest that biologically active lipid mediators in ML exosomes may be involved in the inflammatory response after intestinal ischemia. Level of evidence Not applicable (Basic Science paper).

Intestinal ischemia-reperfusion injury leads to multiple organ injuries via gut-derived mediators following severe injury. Growing evidence suggests that exosomes secreted from intestinal epithelial cells are heavily involved in the development of systemic inflammation, but a full elucidation of its pathology remains to be completed. To produce an integrated understanding of its pathology, this study aimed to reveal the changes in exosome content after ischemic stimulation. Our result showed (1) the proteins involved in inflammation by catalyzing RNAs were upregulated, (2) hsa-miR-21-5p, hsa-miR-23a-3p, and hsa-miR-30d-5p levels were increased while hsa-miR-124-3p level was decreased, (3) the increase in unsaturated lysophosphatidylcholines levels. These results together with those of previous studies, suggest that lysophosphatidylcholines may activate the NK-κB pathway. The proteins and microRNAs jointly act to disrupt negative feedback, thereby increasing inflammation. Thus, our results clarify part of the mechanism of multi-organ failure after intestinal ischemic recanalization, thereby providing a new target for treatment.

Study objective Although geriatric trauma patients are becoming more common, few large-scale analyses have comprehensively evaluated geriatric-specific characteristics in trauma. This study aims to clarify the age-specific characteristics, outcomes, and predictive accuracy of current trauma scoring systems among geriatric trauma patients. Methods Patients with severe trauma, with an Abbreviated Injury Scale score greater than or equal to 3, and registered in the Japan Trauma Data Bank during 2004 to 2015 were retrospectively reviewed. Age-related differences were assessed for injury mechanism, injured region, anatomic and physiologic severity, and inhospital mortality. The mortality risk was evaluated with multivariate mixed-effect models adjusted for Injury Severity Score, Revised Trauma Score, year of injury, and treating facility. Age-related differences in the accuracy of the Injury Severity Score and Revised Trauma Score for predicting inhospital mortality were evaluated with an area under the receiver operating characteristic curve. Results We identified 127,303 patients, including 67,316 geriatric patients (52.9%) who were aged 60 years or older. The percentage of geriatric patients increased from 31.9% to 59.7% during the study period. The most frequent injury mechanism was ground-level falls (55.2%) and the most frequently injured region was the pelvis and lower extremities (43.7%). Severity-adjusted mixed-effects models revealed a marked age-dependent increase in mortality. Although the Injury Severity Score had similar predictive accuracy among all generations, the accuracy of the Revised Trauma Score decreased with increasing age. Conclusion The characteristics of trauma patients varied widely according to age, and mortality risk increased steadily with increasing age, despite a decrease in anatomic injury severity. The Revised Trauma Score had decreasing predictive accuracy at older ages, suggesting that an alternative measure is needed.

To elucidate the host genetic loci affecting severity of SARS-CoV-2 infection, or Coronavirus disease 2019 (COVID-19), is an emerging issue in the face of the current devastating pandemic. Here, we report a genome-wide association study (GWAS) of COVID-19 in a Japanese population led by the Japan COVID-19 Task Force, as one of the initial discovery GWAS studies performed on a non-European population. Enrolling a total of 2,393 cases and 3,289 controls, we not only replicated previously reported COVID-19 risk variants (e.g., LZTFL1, FOXP4, ABO, and IFNAR2), but also found a variant on 5p35 (rs60200309-A at DOCK2) that was significantly associated with severe COVID-19 in younger (-8 (odds ratio = 2.01, 95% confidence interval = 1.58-2.55). This risk allele was prevalent in East Asians, including Japanese (minor allele frequency [MAF] = 0.097), but rarely found in Europeans. Cross-population Mendelian randomization analysis made a causal inference of a number of complex human traits on COVID-19. In particular, obesity had a significant impact on severe COVID-19. The presence of the population-specific risk allele underscores the need of non-European studies of COVID-19 host genetics.

Background Although the results of previous studies suggested the effectiveness of physician-led prehospital trauma management, it has been uncertain because of the limited number of high-quality studies. Furthermore, the advantage of physician-led prehospital management might have been overestimated due to the shortened prehospital time by helicopter transportation in some studies. The present study aimed to evaluate the effect of physician-led prehospital management independent of prehospital time. Also, subgroup analysis was performed to explore the subpopulation that especially benefit from physician-led prehospital management. Methods This retrospective cohort study analyzed the data of Japan’s nationwide trauma registry. Severe blunt trauma patients, defined by Injury Severity Score (ISS) ≥16, who were transported directly to a hospital between April 2009 and March 2019 were evaluated. In-hospital mortality was compared between groups dichotomized by the occupation of primary prehospital healthcare provider (i.e., physician or paramedic), using 1:4 propensity score-matched analysis. The propensity score was calculated using potential confounders including patient demographics, mechanism of injury, vital signs at the scene of injury, ISS, and total time from injury to hospital arrival. Subpopulations that especially benefit from physician-led prehospital management were explored by assessing interaction effects between physician-led prehospital management and patient characteristics. Results A total of 30,551 patients (physician-led: 2976, paramedic-led: 27,575) were eligible for analysis, of whom 2690 propensity score-matched pairs (physician-led: 2690, paramedic-led: 10,760) were generated and compared. Physician-led group showed significantly decreased in-hospital mortality than paramedic-led group (in-hospital mortality: 387 [14.4%] and 1718 [16.0%]; odds ratio [95% confidence interval] = 0.88 [0.78–1.00], p = 0.044). Patients with age Conclusions Physician-led prehospital trauma management was significantly associated with reduced in-hospital mortality independent of prehospital time. The findings of exploratory subgroup analysis would be useful for the future research to establish efficient dispatch system of physician team.

Background: The comparative effectiveness of physician-led over paramedic-led prehospital trauma management has been inconclusive. Regarding this topic, in some previous studies, the impacts of physician-led prehospital management were affected by the advantage of shortened prehospital time by helicopter transportation. This study aimed to evaluate the effect of physician-led prehospital management independent of prehospital time.Methods: This retrospective cohort study analyzed the data of severe trauma patients who were transported directly to a hospital during 2009–2018 using Japan’s nationwide trauma registry. In-hospital mortality was compared between patients who received physician-led prehospital management and those who received paramedic-led management, using 1:4 propensity score-matched analysis. The propensity score was calculated using information on patient demographics, mechanism of injury, and vital signs at the scene of injury, as well as prehospital transport time. Subgroup analysis was performed to identify patients who were most likely to benefit from physician-led prehospital management.Results: A total of 30,968 patients (physician-led: 3,032, paramedic-led: 27,936) were eligible for analysis, of whom 2,766 propensity score-matched pairs (i.e., physician-led: 2766, paramedic-led: 11,064) were generated and compared. Physician-led pre-hospital trauma management showed significant superiority over paramedic-led prehospital trauma management (in-hospital mortality: 395 [14.3%] and 1785 [16.1%], respectively; odds ratio [95% confidence interval] = 0.87 [0.77–0.97], p = 0.017). In subgroup analysis, cases characterized by patient age Conclusions: The result of a largescale registry-based cohort study showed that physician-led prehospital trauma management was significantly associated with survival benefit independent of prehospital transport time. The findings may provide a basis for future research to assess effective physician-provided treatments in prehospital-field.

Background The effectiveness and indications of open-chest cardiopulmonary resuscitation (OCCPR) have been still debatable. Although current guidelines state that the presence of signs of life (SOL) is an indication for OCCPR, scientific evidence corroborating this recommendation has been scarce. This study aimed to compare the effectiveness of OCCPR to closed-chest cardiopulmonary resuscitation (CCCPR) in severe trauma patients with SOL upon arrival at the emergency department (ED). Methods A retrospective cohort study analyzing data from the Trauma Quality Improvement Program (TQIP) database, a nationwide trauma registry in the USA, between 2010 and 2016 was conducted. Severe trauma patients who had SOL upon arrival at the hospital and received cardiopulmonary resuscitation within the first 6 h of ED admission were identified. Survival to hospital discharge was evaluated using logistic regression analysis, instrumental variable analysis, and propensity score matching analysis adjusting for potential confounders. Results A total of 2682 patients (OCCPR 1032; CCCPR 1650) were evaluated; of those 157 patients (15.2%) in the OCCPR group and 193 patients (11.7%) in the CCCPR group survived. OCCPR was significantly associated with higher survival to hospital discharge in both the logistic regression analysis (adjusted odds ratio [95% confidence interval] = 1.99 [1.42–2.79], p p = 0.021). In the propensity score matching analysis, 531 matched pairs were generated, and the OCCPR group still showed significantly higher survival at hospital discharge (89 patients [16.8%] in the OCCPR group vs 58 patients [10.9%] in the CCCPR group; odds ratio [95% confidence interval] = 1.66 [1.13–2.42], p = 0.009). Conclusions Compared to CCCPR, OCCPR was associated with significantly higher survival at hospital discharge in severe trauma patients with SOL upon ED arrival. Further studies to confirm these results and to assess long-term neurologic outcomes are needed.

Background Exosomes are extracellular vesicles that act as endogenous mediators of the immune response. We have previously shown that exosomes released into mesenteric lymph (ML) following trauma (T)/hemorrhagic shock (HS) induce proinflammatory cytokine production in macrophages and are involved in the pathogenesis of postshock acute lung injury. However, the cellular origin of ML exosomes and their role in the posttrauma immune response remains unclear. We hypothesized that exosomes released from damaged-intestinal epithelial cells contribute to posttrauma immune dysfunction by altering the function of dendritic cells (DCs), key regulators of the adaptive immunity. Methods Male rats underwent cannulation of the femoral artery, jugular vein and ML duct. T/HS was induced by laparotomy and 60 minutes of hemorrhagic shock followed by resuscitation. The ML was collected before (preshock) and after T/HS (post-T/HS) for isolation of exosomes. Surface epitopes of exosomes isolated from ML were assessed by flow cytometry to determine their cellular origin and phenotypic changes. The immunomodulatory effects of ML exosomes on DCs were assessed by Annexin V apoptosis assay, expression of costimulatory molecules, and antigen-presenting capacity to lymphocytes. Results Exosomes isolated from ML highly expressed CD63 (exosome marker) and epithelial cell-specific marker, suggesting their derivation from intestinal epithelial cells. The expression of immunomodulatory molecules, such as major histocompatibility complex class II and Fas ligand on ML exosomes, was significantly increased after T/HS. Coincubation of DCs with exosomes isolated from ML after T/HS increased DC apoptosis twofold compared with preshock ML exosomes. Furthermore, post-T/HS ML exosomes significantly suppressed lipopolysaccharide-mediated expression of CD80 and CD86 on DCs as well as decreased their antigen-presenting capacity to induce lymphocytes proliferation. Conclusion Gut epithelial cells release immunomodulatory exosomes into the ML after T/HS and resuscitation. Mesenteric lymph exosomes may be critical mediators of posttraumatic immunosuppression causing depletion and dysfunction of DCs.

Acute lung injury (ALI) is a common cause of morbidity in patients after severe injury due to dysregulated inflammation, which is believed to be driven by gut-derived inflammatory mediators carried via mesenteric lymph (ML). We have previously demonstrated that nano-sized extracellular vesicles, called exosomes, secreted into ML after trauma/hemorrhagic shock (T/HS) have the potential to activate immune cells in vitro Here, we assess the function of ML exosomes in the development of T/HS-induced ALI and the role of TLR4 in the ML exosome-mediated inflammatory response. ML exosomes isolated from rats subjected to T/HS stimulated NF-κB activation and caused proinflammatory cytokine production in alveolar macrophages. In vivo experiments revealed that intravenous injection of exosomes harvested after T/HS, but not before shock, caused recruitment of inflammatory cells in the lung, increased vascular permeability, and induced histologic ALI in naive mice. The exosome-depleted supernatant of ML had no effect on in vitro and in vivo inflammatory responses. We also demonstrated that both pharmacologic inhibition and genetic knockout of TLR4 completely abolished ML exosome-induced cytokine production in macrophages. Thus, our findings define the critical role of exosomes secreted into ML as a critical mediator of T/HS-induced ALI through macrophage TLR4 activation.-Kojima, M., Gimenes-Junior, J. A., Chan, T. W., Eliceiri, B. P., Baird, A., Costantini, T. W., Coimbra, R. Exosomes in postshock mesenteric lymph are key mediators of acute lung injury triggering the macrophage activation via Toll-like receptor 4.

Previous studies have shown that mesenteric lymph (ML) has a crucial role in driving the systemic inflammatory response after trauma/hemorrhagic shock (T/HS). The specific mediators in the ML that contribute to its biological activity remain unclear despite decades of study. Exosomes are extracellular vesicles that are shed into body fluids such as serum and urine that can mediate intercellular communication. We hypothesized that exosomes are present in the ML after trauma/shock and are responsible for the biological activity of ML.Male rats underwent cannulation of the vessels and mesenteric lymph duct. T/HS was induced by laparotomy and 60 minutes of HS (mean arterial pressure, 35 mmHg), followed by resuscitation. The ML was collected during three distinct time periods (pre-shock, shock, and resuscitation phase) and subsequently separated into exosome and supernatant fractions. Exosomes were characterized by electron microscope, nanoparticle tracking analysis, and immunoblotting. The biological activity of exosomes and supernatant of ML were characterized using a monocyte NF-κB reporter assay and by measuring macrophage intracellular TNF-α production.Exosomes were identified in ML by size and expression of the exosome markers CD63 and HSP70. The number of exosomes present in the ML was 2-fold increased during shock and 4-fold decreased in resuscitation phase compared to pre-shock. However, biological activity of exosomes isolated during the resuscitation phase was markedly increased and caused an 8-fold increase in monocyte NF-κB activation compared to supernatant. Macrophage TNF-α production was also increased after exposure to exosomes harvested in the resuscitation phase. The ML supernatant fraction had no effect on TNF-α production during any phase.Our findings show that exosomes, and not the liquid fraction of ML, are the major component triggering inflammatory responses in monocytes and macrophages after experimental T/HS.

Nonocclusive mesenteric ischemia (NOMI) is an acute and life-threatening gastrointestinal disorder, requiring rapid therapeutic intervention for ischemic bowel. However, its rapid detection remains challenging. This retrospective, observational study was aimed at comparing the diagnostic accuracy for NOMI in models of biomarkers, including intestinal fatty acid-binding protein (I-FABP), and clinical findings.All consecutive patients who presented to the emergency department of the study hospital with suspected NOMI were prospectively enrolled. Receiver operating characteristic analysis compared the diagnostic accuracy of I-FABP with traditional biomarkers (white blood cell count, C-reactive protein, lactate, creatine kinase, and D-dimer) alone and in combination with the baseline model established from clinical findings.Of 96 patients with suspected NOMI, 25 (26.0%) were clinically diagnosed with NOMI. In-hospital mortality was higher in patients with NOMI than those with other conditions (56.0% vs. 4.2%, p0.001). Receiver operating characteristic analyses revealed that the I-FABP model had the highest area under the curve (0.805) in the diagnosis of NOMI, compared with other biomarkers. The diagnostic model of clinical findings including age, cardiovascular disease history, undergoing hemodialysis, hypotension, and consciousness disturbance in combination with I-FABP showed the best discrimination (area under the curve, 0.883), compared with other biomarkers. The bootstrap optimism estimate showed the lowest discrimination among the other models with other biomarkers (0.006).The usefulness of I-FABP for final diagnosis of NOMI in patients with clinically suspected NOMI at the emergency department was internally validated. Further external validation study is warranted.Diagnostic test, level III.

BACKGROUND Phospholipase A2 (PLA2) is associated with a variety of inflammatory processes related to polymorphonuclear neutrophil (PMN)-endothelial cell interactions. However, the cellular and molecular mechanisms underlying the interactions and the causative isoform(s) of PLA2 remain elusive. In addition, we recently showed that calcium-independent PLA2γ (iPLA2γ), but not cytosolic PLA2 (cPLA2), is responsible for the cytotoxic functions of human PMN including respiratory bursts, degranulation, and chemotaxis. We therefore hypothesized that iPLA2γ is a prerequisite for the PMN recruitment cascade into the site of inflammation. The aim of this study was to elucidate the roles of the three major phospholipases A2, iPLA2, cPLA2 and secretory PLA2, in leukocyte rolling and adherence and in the surface expression of β2-integrins in vivo and in vitro in response to well-defined stimuli. METHODS Male Wistar rats were pretreated with PLA2 inhibitors selective for iPLA2β, iPLA2γ, cPLA2, or secretory PLA2. Leukocyte rolling/adherence in the mesenteric venules superfused with platelet-activating factor (PAF) were quantified by intravital microscopy. Furthermore, isolated human PMNs or whole blood were incubated with each PLA2 inhibitor and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or PAF. PMN adherence was assessed by counting cells bound to purified fibrinogen, and the surface expression of lymphocyte function-associated antigen 1 and macrophage antigen 1 (Mac-1) was measured by flow cytometry. RESULTS The iPLA2γ-specific inhibitor almost completely inhibited the fMLP/PAF-induced leukocyte adherence in vivo and in vitro and also decreased the fMLP/PAF-stimulated surface expression of Mac-1 by 60% and 95%, respectively. In contrast, the other inhibitors did not affect these cellular functions. CONCLUSION iPLA2γ seems to be involved in leukocyte/PMN adherence in vivo and in vitro as well as in the up-regulation of Mac-1 in vitro in response to PAF/fMLP. This enzyme is therefore likely to be a major regulator in the PMN recruitment cascade.

The role of calcium-independent phospholipase A2 (iPLA2), a component of the three major PLA2 families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA2-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA2 is unidentified. We also demonstrated that the iPLA2γ-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA2γ may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA2 (cPLA2), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA2γ regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A2, iPLA2, and cPLA2, in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA.Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2β, iPLA2γ, or cPLA2 and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system.The iPLA2γ-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA2-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA2γ, but not iPLA2β or cPLA2, completely inhibited the platelet activating factor/fMLP-induced chemotaxis.iPLA2 is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA2 inhibitor suggests that AA may not be relevant to these cytotoxic functions.