Plasma Proteomics Profile-Based Comparison of Torso Versus Brain Injury: A Prospective Cohort Study.

INTRODUCTION: Trauma-related deaths and post-traumatic sequelae are a global health concern, necessitating a deeper understanding of the pathophysiology to advance trauma therapy. Proteomics offers insights into identifying and analyzing plasma proteins associated with trauma and inflammatory conditions; however, current proteomic methods have limitations in accurately measuring low-abundance plasma proteins. This study compared plasma proteomics profiles of patients from different acute trauma subgroups to identify new therapeutic targets and devise better strategies for personalized medicine. METHODS: This prospective observational single-center cohort study was conducted between August 2020 and September 2021 in the intensive care unit of Osaka University Hospital in Japan. Enrolling 59 consecutive patients with blunt trauma, we meticulously analyzed plasma proteomics profiles in participants with torso or head trauma, comparing them with those of controls (mild trauma). Using the Olink Explore 3072® instrument, we identified five endotypes (α-ε) via unsupervised hierarchical clustering. RESULTS: The median time from injury to blood collection was 47 minutes [interquartile range: 36-64 minutes]. The torso trauma subgroup exhibited 26 unique proteins with significantly altered expression, while the head trauma subgroup showed 68 unique proteins with no overlap between the two. The identified endotypes included α (torso trauma, n = 8), ß (young patients with brain injury, n = 5), γ (severe brain injury post-surgery, n = 8), δ (torso or brain trauma with mild hyperfibrinolysis, n = 18), and ε (minor trauma, n = 20). Patients with torso trauma showed changes in blood pressure, smooth muscle adaptation, hypermetabolism, and hypoxemia. Patients with traumatic brain injury had dysregulated blood coagulation and altered nerves regeneration and differentiation. CONCLUSIONS: This study identified unique plasma protein expression patterns in patients with torso trauma and traumatic brain injury, helping categorize five distinct endotypes. Our findings may offer new insights for clinicians, highlighting potential strategies for personalized medicine and improved trauma-related care. LEVEL OF EVIDENCE: Prospective Cohort Study, Level III.

T cell dysfunction in elderly ARDS patients based on miRNA and mRNA integration analysis.

Background: Acute respiratory distress syndrome (ARDS) is respiratory failure that commonly occurs in critically ill patients, and the molecular mechanisms underlying its pathogenesis and severity are poorly understood. We evaluated mRNA and miRNA in patients with ARDS and elucidated the pathogenesis of ARDS after performing mRNA and miRNA integration analysis. Methods: In this single-center, prospective, observational clinical study of patients with ARDS, peripheral blood of each patient was collected within 24 hours of admission. Sequencing of mRNA and miRNA was performed using whole blood from the ARDS patients and healthy donors. Results: Thirty-four ARDS patients were compared with 15 healthy donors. Compared with the healthy donors, 1233 mRNAs and 6 miRNAs were upregulated and 1580 mRNAs and 13 miRNAs were downregulated in the ARDS patients. For both mRNA and miRNA-targeted mRNA, canonical pathway analysis showed that programmed death-1 (PD-1) and programmed cell death ligand 1 (PD-L1) cancer immunotherapy pathway was most activated and the Th2 pathway was most suppressed. For mRNA, the Th1 pathway was most suppressed. miR-149-3p and several miRNAs were identified as upstream regulators. Conclusion: miRNAs regulated the PD-1 and PD-L1 cancer immunotherapy pathway and Th2 pathway through miRNA interference action of mRNA. Integrated analysis of mRNAs and miRNAs showed that T cells were dysfunctional in ARDS patients.

Relationship between extravascular leakage and clinical outcome on computed tomography of isolated traumatic brain injury.

Aim: This study investigated whether contrast extravasation on computed tomography (CT) angiography in patients with traumatic brain injury (TBI) is associated with death or surgical procedures. Methods: Patients over 18 years old, directly brought in by ambulance with an isolated head injury and confirmed to have acute intracranial hemorrhage on a CT scan upon admission between 2010 and 2020, were included. The primary outcome was mortality, and the secondary outcome was neurosurgical procedures performed from admission to discharge from the intensive care unit. Multivariable logistic regression analyses were performed to evaluate the association between these outcomes and contrast extravasation. Results: The analysis included 188 patients with a median age of 65 years, 123 men (65.4%), 34 deaths (18.1%), and 91 surgeries (48.4%). Among the 66 patients with contrast extravasation, 22 (33.3%) died and 47 (71.2%) required surgery. Among the 122 patients with no contrast extravasation, 12 (9.8%) died, and 44 (36.1%) required surgery. The presence or absence of extravascular leakage was associated with death (odds ratio, 3.6 [95% CI: 1.2-12.2]) and surgery (odds ratio, 7.6 [95% CI: 2.5-22.7]). Conclusion: Contrast extravasation was associated with mortality and performance of surgery in patients with an isolated head injury.

Classification of patients with COVID-19 by blood RNA endotype: a prospective cohort study.

IMPORTANCE: In this study, whole-blood RNAs (prolactin and toll-like receptor 3) involved in the prognosis of patients with COVID-19 were identified. The RNA endotypes classified by these important RNAs highlight the possibility of stratifying the COVID-19 patient population and the need for targeted therapy based on these phenotypes.

Complement factor D targeting protects endotheliopathy in organoid and monkey models of COVID-19.

COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes.

Importance of microRNAs by mRNA-microRNA integration analysis in acute ischemic stroke patients.

OBJECTS: The roles of mRNA and microRNA (miRNA) are well known in many diseases, including ischemic stroke; thus, integration analysis using mRNA and miRNA is important to elucidate pathogenesis. However, their contribution, especially that of miRNA-targeted mRNA, to the severity of acute ischemic stroke remains unclear. Therefore, we examined mRNA and miRNA integration analysis targeted for acute ischemic stroke to clarify the pathway related to acute stroke severity. MATERIAL AND METHODS: We performed Ingenuity Pathway Analysis (IPA) using RNA extracted from the whole blood of four healthy controls, six minor acute ischemic stroke patients (MS; National Institutes of Health Stroke Scale [NIHSS] < 8), and six severe acute ischemic stroke patients (SS; NIHSS ≥ 8) on admission. mRNA and miRNA were measured using RNA sequencing and RNA expression variation; canonical pathway analysis (CPA) and upstream regulator analyses were performed. RESULTS: Acute ischemic stroke patients demonstrated different RNA expressions to healthy controls. Compared to MS patients, in the SS patients, 1222 mRNA, 96 miRNA, and 935 miRNA-targeted mRNA expressions were identified among differentially expressed RNA expressions (p<0.05, |log2 fold change| >1.1). CPA by IPA using mRNAs or miRNA-targeted mRNAs showed that macrophage-stimulating protein (MSP)-recepteur d'origine nantais (RON) signaling was mostly activated in SS patients compared to in MS patients. In addition, upstream regulator analysis in IPA showed that most mRNAs located upstream are miRNAs. CONCLUSIONS: In severe acute stroke, integration of mRNA and microRNA analysis showed activated MSP-RON signaling in macrophages, and multiple miRNAs comprehensively controlled the overall pathophysiology of stroke.

Whole-blood ribonucleic acid sequencing analysis in methemoglobinemia: a case report.

INTRODUCTION: Methemoglobinemia is a condition in which methemoglobin is increased and the oxygen carrying capacity of tissues is decreased, causing a lack of oxygen to the whole body. RNA (ribonucleic acid) sequencing technologies have made it possible to systematically examine how the human transcriptome responds to invasive pathologies. To our knowledge, no previous studies have reported the results of RNA sequencing in a patient with methemoglobinemia. We describe the analysis of RNAs from the whole blood of a patient with methemoglobinemia. CASE PRESENTATION: A 31-year-old Japanese man was brought to our hospital with symptoms of dyspnea due to inhalation of gas from an acetic acid phosphonitrate storage tank at a factory. The nitrogen oxide concentration measured around the storage tank was over 2500 ppm, and he witnessed orange-brown smoke at that time. After entering the area and taking a few breaths, he suddenly became unwell, with dyspnea and numbness in his extremities. He was evacuated from the area within a few minutes, at which time he was suffering from whole-body cyanosis and was still aware of the above symptoms. On arrival at the hospital, his respiration rate was 18 breaths/minute, and his SpO2 ranged from 80% to 85% on 15 L/minute of oxygen by mask (2.5 hours postexposure). Arterial blood gas testing revealed a methemoglobin level of 23.1%. After the administration of methylene blue, the patient's methemoglobin level normalized and his symptoms improved. Chest X-ray and chest computed tomography showed no evidence of pulmonary edema or interstitial pneumonia, and no other abnormal findings were observed. RNA sequencing was performed on the blood samples obtained at the time of the visit, with the blood sample collected on day 5 used as a control. To our knowledge, the present study is the first to describe the analysis of RNAs from the whole blood of a patient with methemoglobinemia. The RNA sequencing analysis showed that an activated "hydrogen peroxide catabolic process" may be associated with the pathogenesis of methemoglobinemia. CONCLUSION: The results reported in the present study may explain the pathogenesis of methemoglobinemia.

Combination of WFDC2, CHI3L1, and KRT19 in Plasma Defines a Clinically Useful Molecular Phenotype Associated with Prognosis in Critically Ill COVID-19 Patients.

BACKGROUND: COVID-19 is now a common disease, but its pathogenesis remains unknown. Blood circulating proteins reflect host defenses against COVID-19. We investigated whether evaluation of longitudinal blood proteomics for COVID-19 and merging with clinical information would allow elucidation of its pathogenesis and develop a useful clinical phenotype. METHODS: To achieve the first goal (determining key proteins), we derived plasma proteins related to disease severity by using a first discovery cohort. We then assessed the association of the derived proteins with clinical outcome in a second discovery cohort. Finally, the candidates were validated by enzyme-linked immunosorbent assay in a validation cohort to determine key proteins. For the second goal (understanding the associations of the clinical phenotypes with 28-day mortality and clinical outcome), we assessed the associations between clinical phenotypes derived by latent cluster analysis with the key proteins and 28-day mortality and clinical outcome. RESULTS: We identified four key proteins (WFDC2, GDF15, CHI3L1, and KRT19) involved in critical pathogenesis from the three different cohorts. These key proteins were related to the function of cell adhesion and not immune response. Considering the multicollinearity, three clinical phenotypes based on WFDC2, CHI3L1, and KRT19 were identified that were associated with mortality and clinical outcome. CONCLUSION: The use of these easily measured key proteins offered new insight into the pathogenesis of COVID-19 and could be useful in a potential clinical application.

Development of clinical phenotypes and biological profiles via proteomic analysis of trauma patients.

BACKGROUND: Trauma is a heterogeneous condition, and specific clinical phenotypes may identify target populations that could benefit from certain treatment strategies. In this retrospective study, we determined clinical phenotypes and identified new target populations of trauma patients and their treatment strategies. METHODS: We retrospectively analyzed datasets from the Japan Trauma Data Bank and determined trauma death clinical phenotypes using statistical machine learning techniques and evaluation of biological profiles. RESULTS: The analysis included 71,038 blunt trauma patients [median age, 63 (interquartile range [IQR], 40-78) years; 45,479 (64.0%) males; median Injury Severity Score, 13 (IQR, 9-20)], and the derivation and validation cohorts included 42,780 (60.2%) and 28,258 (39.8%) patients, respectively. Of eight derived phenotypes (D-1-D-8), D-8 (n = 2178) had the highest mortality (48.6%) with characteristic severely disturbed consciousness and was further divided into four phenotypes: D-8α, multiple trauma in the young (n = 464); D-8ß, head trauma with lower body temperature (n = 178); D-8γ, severe head injury in the elderly (n = 957); and D-8δ, multiple trauma, with higher predicted mortality than actual mortality (n = 579). Phenotype distributions were comparable in the validation cohort. Biological profile analysis of 90 trauma patients revealed that D-8 exhibited excessive inflammation, including enhanced acute inflammatory response, dysregulated complement activation pathways, and impaired coagulation, including downregulated coagulation and platelet degranulation pathways, compared with other phenotypes. CONCLUSIONS: We identified clinical phenotypes with high mortality, and the evaluation of the molecular pathogenesis underlying these clinical phenotypes suggests that lethal trauma may involve excessive inflammation and coagulation disorders.

Resistin Associated With Cytokines and Endothelial Cell Adhesion Molecules Is Related to Worse Outcome in COVID-19.

Introduction: Resistin is reported to form a cytokine network and cause endothelial damage. The pathogenesis of coronavirus disease 2019 (COVID-19) remains unknown, but the association between cytokine storm and endothelial damage is crucial. This study aimed to evaluate resistin in COVID-19 pathogenesis compared with sepsis. Materials and Methods: First, we evaluated the association of plasma resistin levels and disease severity and clinical outcome in two large cohorts: a publicly available cohort including 306 COVID-19 patients in the United States (MGH cohort) and our original cohort including only intubated 113 patients in Japan (Osaka cohort 1). Second, to understand pathogenesis, we evaluate resistin, cytokines and endothelial cell adhesion molecules in COVID-19 compared with sepsis. Blood samples were collected from 62 ICU-treated COVID-19 patients and 38 sepsis patients on day 1 (day of ICU admission), days 2-3, days 6-8, and from 18 healthy controls (Osaka cohort 2). The plasma resistin, inflammatory cytokines (IL-6, IL-8, MCP-1 and IL-10) and endothelial cell adhesion molecules (ICAM-1 and VCAM-1) were compared between patients and control. Correlations among resistin, inflammatory cytokines and endothelial cell adhesion molecules were evaluated in COVID-19 and sepsis. Results: In the MGH cohort, the day 1 resistin levels were associated with disease severity score. The non-survivors showed significantly greater resistin levels than survivors on days 1, 4 and 8. In the Osaka cohort 1, 28-day non-survivors showed significantly higher resistin levels than 28-day survivors on days 6-8. Patients with late recovery (defined as the day of weaning off mechanical ventilation >12 or death) had significantly higher resistin levels than those with early recovery on day 1 and days 6-8. In the Osaka cohort 2, plasma resistin levels were elevated in COVID-19 and sepsis patients compared to controls at all measurement points and were associated with inflammatory cytokines and endothelial cell adhesion molecules. Conclusion: Resistin was elevated in COVID-19 patients and was associated with cytokines and endothelial cell adhesion molecules. Higher resistin levels were related to worse outcome.

Significance of interferon signaling based on mRNA-microRNA integration and plasma protein analyses in critically ill COVID-19 patients.

We evaluated mRNA and miRNA in COVID-19 patients and elucidated the pathogenesis of COVID-19, including protein profiles, following mRNA and miRNA integration analysis. mRNA and miRNA sequencing was done on admission with whole blood of 5 and 16 healthy controls (HCs) and 10 and 31 critically ill COVID-19 patients (derivation and validation cohorts, respectively). Interferon (IFN)-α2, IFN-ß, IFN-γ, interleukin-27, and IFN-λ1 were measured in COVID-19 patients on admission (day 1, 181 critical/22 non-critical patients) and days 6-8 (168 critical patients) and in 19 HCs. In the derivation cohort, 3,488 mRNA and 31 miRNA expressions were identified among differentially expressed RNA expressions in the patients versus those in HCs, and 2,945 mRNA and 32 miRNA expressions in the validation cohort. Canonical pathway analysis showed the IFN signaling pathway to be most activated. The IFN-ß plasma level was elevated in line with increased severity compared with HCs, as were IFN-ß downstream proteins, such as interleukin-27. IFN-λ1 was higher in non-critically ill patients versus HCs but lower in critical than non-critical patients. Integration of mRNA and miRNA analysis showed activated IFN signaling. Plasma IFN protein profile revealed that IFN-ß (type I) and IFN-λ1 (type III) played important roles in COVID-19 disease progression.

Growth Differentiation Factor-15 Correlates with Mortality and Severity in Severe Burns.

BACKGROUND: Growth differentiation factor-15 (GDF-15) is expressed in almost all tissues of the body and is necessary for the body's defense response to stress such as inflammation. It has been reported to be associated with incidence and mortality in many diseases, including systemic inflammatory response syndromes. There are no reports on GDF-15 in burns. The purpose of this study was to investigate the trend of GDF-15 in blood in patients with severe burns and to determine its relationship with severity and mortality. METHODS: This was a retrospective, observational, single-center study. The level of GDF-15 in the blood was measured and compared with clinical parameters, including prognosis. Time points for sample collection were the day of injury, 4 days after injury, and 1 week after injury. RESULTS: Eighty-three patients were enrolled in the study. At all time points, GDF-15 levels in the nonsurvivor group were significantly higher than those in the survivor group. In the analysis using the ROC curve for 28-day survival, the AUC of the GDF-15 value on the day of injury was 0.798, which was higher than those of % total body surface area, burn index, and Sequential Organ Failure Assessment (SOFA) score. GDF-15 levels correlated positively with SOFA score, and the relationship became stronger along with the time course of severe burn. CONCLUSIONS: In the acute phase of severe burn, GDF-15 levels were associated with mortality and SOFA scores.

Association of Extravascular Leakage on Computed Tomography Angiography with Fibrinogen Levels at Admission in Patients with Traumatic Brain Injury.

Extravascular leakage on computed tomography (CT) angiography in patients with traumatic brain injury (TBI) is associated with hematoma expansion, functional prognosis, subsequent surgery, and death. Fresh frozen plasma (FFP) administration is often necessary to treat coagulation disorders associated with TBI. This study aimed to determine the relationship between the presence of extravascular leakage on contrast-enhanced head CT, fibrinogen level at admission, and FFP administration in patients with TBI. The medical records of patients with TBI ≥18 years of age referred to our hospital between January 2010 and December 2020 were examined retrospectively. Patients who underwent contrast-enhanced CT immediately after admission were selected, and the presence or absence of extravascular leakage, fibrinogen level at admission, and percentage of patients who required FFP administration within 24 h of admission were examined; 172 patients were included. Multi-variable linear regression analysis was performed to determine the effects of contrast extravasation on fibrinogen levels at admission and was adjusted for age, sex, systolic blood pressure, time from injury to admission, Marshall CT score, Glasgow Coma Scale score at admission, Injury Severity Score, and need for emergency surgery; the regression coefficient was -19.8. The effect of extravasation on FFP administration within 24 h of admission was analyzed using logistic regression while adjusting for age, systolic blood pressure, Marshall CT score, need for emergency surgery, and fibrinogen level at admission. The odds ratio of contrast extravasation was 7.08 after adjustment. Extravascular leakage is associated with fibrinogen levels at admission and FFP administration within 24 h of admission.

Original experimental rat model of blast-induced mild traumatic brain injury: a pilot study.

OBJECTIVE: Diagnosing blast-induced mild traumatic brain injury (mTBI) is difficult due to minimal imaging findings. This study aimed to establish a rat model of behavioral abnormality caused by blast-induced mTBI and detect new findings for therapeutic intervention. METHODS: We used a bench-top blast wave generator with the blast wave exiting through a 20-mm I.D. nozzle aimed at the focused target. The blast wave was directed at the head of male Wistar rats under general anesthesia positioned prone 2.5 cm below the nozzle. Peak shock wave pressure was 646.2 ± 70.3 kPa. RESULTS: After blast injury, mTBI rats did not show the findings of brain hemorrhage or contusion macroscopically and on hematoxylin-eosin-stained frozen sections but did show anorexia and weight loss in the early post-injury phase. Behavioral experiments revealed short-term memory impairment at 2 weeks and depression-like behavior at 2 and 6 weeks. Diffusion-weighted ex vivo MRI showed high-intensity areas in layers of the bilateral hippocampus. Immunohistochemical analysis revealed accumulation of reactive microglia and GFAP-positive astrocytes in the same region and loss of NeuN-positive neurons in the hippocampal pyramidal cell layer. CONCLUSIONS: This model can reflect the pathophysiology of blast-induced mTBI and could potentially be used to develop therapeutic interventions in the future.

Cytokine Elevation in Severe COVID-19 From Longitudinal Proteomics Analysis: Comparison With Sepsis.

Introduction: Coronavirus disease 2019 (COVID-19) is a new viral disease. Uncontrolled inflammation called "cytokine storm" is reported to contribute to disease pathogenesis as well as sepsis. We aimed to identify cytokines related to the pathogenesis of COVID-19 through a proteomics analysis of 1463 plasma proteins, validate these cytokines, and compare them with sepsis. Materials and Methods: In a derivation cohort of 306 patients with COVID-19, 1463 unique plasma proteins were measured on days 1, 4, and 8. Cytokines associated with disease severity and prognosis were derived. In a validation cohort of 62 COVID-19 patients and 38 sepsis patients treated in the intensive care unit [ICU], these derived cytokines were measured on days 1 (day of ICU admission), 2-3, and 6-8 (maximum: 3 time points/patient). Derived cytokines were compared with healthy controls and between COVID-19 and sepsis patients, and the associations with prognosis were evaluated. The time to wean off mechanical ventilation (MV) was evaluated only for COVID-19. Results: IL-6, amphiregulin, and growth differentiation factor (GDF)-15 were associated with disease severity and prognosis in the derivation cohort. In the validation cohort, IL-6 and GDF-15 were elevated in COVID-19 and sepsis on day 1, and the levels of these cytokines were higher in sepsis than in COVID-19. IL-6 and GDF-15 were associated with prognosis in sepsis. Cox proportional hazards model with time as a dependent covariate showed a significant relationship between plasma GDF-15 level and time to wean off MV (hazard ratio, 0.549 [95% confidence level, 0.382-0.789]). The GDF-15 level at ICU admission predicted late recovery. Conclusion: GDF-15 and IL-6 derived from proteomics analysis were related with disease severity of COVID-19. Their values were higher in sepsis than in COVID-19 and were associated with prognosis in sepsis. In COVID-19 patients treated in the ICU, GDF-15 was associated with the time to wean off MV and better predicted late recovery.