The plasma proteome differentiates the multisystem inflammatory syndrome in children (MIS-C) from children with SARS-CoV-2 negative sepsis.

BACKGROUND: The Multi-System Inflammatory Syndrome in Children (MIS-C) can develop several weeks after SARS-CoV-2 infection and requires a distinct treatment protocol. Distinguishing MIS-C from SARS-CoV-2 negative sepsis (SCNS) patients is important to quickly institute the correct therapies. We performed targeted proteomics and machine learning analysis to identify novel plasma proteins of MIS-C for early disease recognition. METHODS: A case-control study comparing the expression of 2,870 unique blood proteins in MIS-C versus SCNS patients, measured using proximity extension assays. The 2,870 proteins were reduced in number with either feature selection alone or with a prior COMBAT-Seq batch effect adjustment. The leading proteins were correlated with demographic and clinical variables. Organ system and cell type expression patterns were analyzed with Natural Language Processing (NLP). RESULTS: The cohorts were well-balanced for age and sex. Of the 2,870 unique blood proteins, 58 proteins were identified with feature selection (FDR-adjusted P < 0.005, P < 0.0001; accuracy = 0.96, AUC = 1.00, F1 = 0.95), and 15 proteins were identified with a COMBAT-Seq batch effect adjusted feature selection (FDR-adjusted P < 0.05, P < 0.0001; accuracy = 0.92, AUC = 1.00, F1 = 0.89). All of the latter 15 proteins were present in the former 58-protein model. Several proteins were correlated with illness severity scores, length of stay, and interventions (LTA4H, PTN, PPBP, and EGF; P < 0.001). NLP analysis highlighted the multi-system nature of MIS-C, with the 58-protein set expressed in all organ systems; the highest levels of expression were found in the digestive system. The cell types most involved included leukocytes not yet determined, lymphocytes, macrophages, and platelets. CONCLUSIONS: The plasma proteome of MIS-C patients was distinct from that of SCNS. The key proteins demonstrated expression in all organ systems and most cell types. The unique proteomic signature identified in MIS-C patients could aid future diagnostic and therapeutic advancements, as well as predict hospital length of stays, interventions, and mortality risks.

Pediatric sepsis inflammatory blood biomarkers that correlate with clinical variables and severity of illness scores.

BACKGROUND: Sepsis is a dysregulated systemic inflammatory response triggered by infection, resulting in organ dysfunction. A major challenge in clinical pediatrics is to identify sepsis early and then quickly intervene to reduce morbidity and mortality. As blood biomarkers hold promise as early sepsis diagnostic tools, we aimed to measure a large number of blood inflammatory biomarkers from pediatric sepsis patients to determine their predictive ability, as well as their correlations with clinical variables and illness severity scores. METHODS: Pediatric patients that met sepsis criteria were enrolled, and clinical data and blood samples were collected. Fifty-eight inflammatory plasma biomarker concentrations were determined using immunoassays. The data were analyzed with both conventional statistics and machine learning. RESULTS: Twenty sepsis patients were enrolled (median age 13 years), with infectious pathogens identified in 75%. Vasopressors were administered to 85% of patients, while 55% received invasive ventilation and 20% were ventilated non-invasively. A total of 24 inflammatory biomarkers were significantly different between sepsis patients and age/sex-matched healthy controls. Nine biomarkers (IL-6, IL-8, MCP-1, M-CSF, IL-1RA, hyaluronan, HSP70, MMP3, and MMP10) yielded AUC parameters > 0.9 (95% CIs: 0.837-1.000; p < 0.001). Boruta feature reduction yielded 6 critical biomarkers with their relative importance: IL-8 (12.2%), MCP-1 (11.6%), HSP70 (11.6%), hyaluronan (11.5%), M-CSF (11.5%), and IL-6 (11.5%); combinations of 2 biomarkers yielded AUC values of 1.00 (95% CI: 1.00-1.00; p < 0.001). Specific biomarkers strongly correlated with illness severity scoring, as well as other clinical variables. IL-3 specifically distinguished bacterial versus viral infection (p < 0.005). CONCLUSIONS: Specific inflammatory biomarkers were identified as markers of pediatric sepsis and strongly correlated to both clinical variables and sepsis severity.

Post-Intensive Care Sequelae in Pediatrics-Results of an Early Rehabilitation Implementation Study.

OBJECTIVES: To compare post-PICU discharge functioning, health-related quality of life (HRQL), and parental stress before and after the implementation of an early rehabilitation bundle. DESIGN AND SETTING: Prospective cohort substudy within an early rehabilitation implementation program, conducted at the PICUs at McMaster Children's Hospital and London Health Sciences, London, Ontario, Canada. INTERVENTIONS: A bundle consisting of: 1) analgesia-first sedation; 2) delirium monitoring and prevention; and 3) early mobilization. Patients with an anticipated 48-hour PICU length of stay were approached for consent to participate. PATIENTS: Critically ill children with an anticipated 48-hour PICU length of stay were approached for consent to participate. MEASUREMENTS AND MAIN RESULTS: Patient-/proxy-reported outcome measures were assessed at baseline, PICU discharge, and 1 and 3 months post-PICU discharge using: 1) Pediatric Evaluation of Disability Inventory Computer Adaptive Test to assess physical, social, cognitive, and responsibility/caregiver domains of functioning; 2) KIDSCREEN to assess HRQL; and 3) the Pediatric Inventory for Parents to assess caregiver stress. A total of 117 participants were enrolled. Patient demographic characteristics were similar in the pre- and post-intervention groups. Following bundle implementation, 30 of 47 respondents (63.8%) experienced functional decline and 18 of 45 (40%) experienced low HRQL at PICU discharge. Eighteen of 36 (50%) at 1 month and 14 of 38 (36.8%) at 3 months experienced either persistent functional decline and/or low HRQL; 2.8% and 2.6% at 1- and 3-month follow-up, respectively, experienced both persistent functional decline and low HRQL. There were no significant differences in the rates of persistent functional decline, low HRQL, or caregiver stress scores post-bundle compared with pre-rehabilitation bundle implementation. CONCLUSIONS: We were unable to adequately determine the efficacy of a rehabilitation bundle on patient-centered outcomes as this substudy was not powered for these outcomes. Our results did reveal that persistent low functioning is common in PICU survivors, more common than low HRQL, while experiencing both functional decline and low HRQL was uncommon.

Early Rehabilitation in Critically ill Children: A Two Center Implementation Study.

OBJECTIVES: To implement an early rehabilitation bundle in two Canadian PICUs. DESIGN AND SETTING: Implementation study in the PICUs at McMaster Children's Hospital (site 1) and London Health Sciences (site 2). PATIENTS: All children under 18 years old admitted to the PICU were eligible for the intervention. INTERVENTIONS: A bundle consisting of: 1) analgesia-first sedation; 2) delirium monitoring and prevention; and 3) early mobilization. MEASUREMENTS AND MAIN RESULTS: Primary outcomes were the duration of implementation, bundle compliance, process of care, safety, and the factors influencing implementation. Secondary endpoints were the impact of the bundle on clinical outcomes such as pain, delirium, iatrogenic withdrawal, ventilator-free days, length of stay, and mortality. Implementation occurred over 26 months (August 2018 to October 2020). Data were collected on 1,036 patients representing 4,065 patient days. Bundle compliance was optimized within 6 months of roll-out. Goal setting for mobilization and level of arousal improved significantly (p < 0.01). Benzodiazepine, opioid, and dexmedetomidine use decreased in site 1 by 23.2% (95% CI, 30.8-15.5%), 26.1% (95% CI, 34.8-17.4%), and 9.2% (95% CI, 18.2-0.2%) patient exposure days, respectively, while at site 2, only dexmedetomidine exposure decreased significantly by 10.5% patient days (95% CI, 19.8-1.1%). Patient comfort, safety, and nursing workload were not adversely affected. There was no significant impact of the bundle on the rate of delirium, ventilator-free days, length of PICU stay, or mortality. Key facilitators to implementation included institutional support, unit-wide practice guidelines, dedicated PICU educators, easily accessible resources, and family engagement. CONCLUSIONS: A rehabilitation bundle can improve processes of care and reduce patient sedative exposure without increasing patient discomfort, nursing workload, or harm. We did not observe an impact on short-term clinical outcomes. The efficacy of a PICU-rehabilitation bundle requires ongoing study. Lessons learned in this study provide evidence to inform rehabilitation implementation in the PICU setting.

A novel multiplex biomarker panel for profiling human acute and chronic kidney disease.

Acute and chronic kidney disease continues to confer significant morbidity and mortality in the clinical setting. Despite high prevalence of these conditions, few validated biomarkers exist to predict kidney dysfunction. In this study, we utilized a novel kidney multiplex panel to measure 21 proteins in plasma and urine to characterize the spectrum of biomarker profiles in kidney disease. Blood and urine samples were obtained from age-/sex-matched healthy control subjects (HC), critically-ill COVID-19 patients with acute kidney injury (AKI), and patients with chronic or end-stage kidney disease (CKD/ESKD). Biomarkers were measured with a kidney multiplex panel, and results analyzed with conventional statistics and machine learning. Correlations were examined between biomarkers and patient clinical and laboratory variables. Median AKI subject age was 65.5 (IQR 58.5-73.0) and median CKD/ESKD age was 65.0 (IQR 50.0-71.5). Of the CKD/ESKD patients, 76.1% were on hemodialysis, 14.3% of patients had kidney transplant, and 9.5% had CKD without kidney replacement therapy. In plasma, 19 proteins were significantly different in titer between the HC versus AKI versus CKD/ESKD groups, while NAG and RBP4 were unchanged. TIMP-1 (PPV 1.0, NPV 1.0), best distinguished AKI from HC, and TFF3 (PPV 0.99, NPV 0.89) best distinguished CKD/ESKD from HC. In urine, 18 proteins were significantly different between groups except Calbindin, Osteopontin and TIMP-1. Osteoactivin (PPV 0.95, NPV 0.95) best distinguished AKI from HC, and ß2-microglobulin (PPV 0.96, NPV 0.78) best distinguished CKD/ESKD from HC. A variety of correlations were noted between patient variables and either plasma or urine biomarkers. Using a novel kidney multiplex biomarker panel, together with conventional statistics and machine learning, we identified unique biomarker profiles in the plasma and urine of patients with AKI and CKD/ESKD. We demonstrated correlations between biomarker profiles and patient clinical variables. Our exploratory study provides biomarker data for future hypothesis driven research on kidney disease.

Pivoting injury prevention efforts during a pandemic: results of an international survey.

BACKGROUND: The COVID-19 a pandemic changed the world. Public health directives to socially distance with stay-at-home orders altered injury risk factor exposure, resulting injury patterns and conducting injury prevention (IP). The objective of this study was to determine the impact the COVID-19 pandemic on injury and IP at North American trauma centers (TC). RESULTS: Sixty-two responses were received from pediatric (44%), adult (11%), and combined (31%) TC, from 22 American states, 5 Canadian provinces and Australia. The majority (91%) of programs targeted age groups from birth to 15 years old. Nearly one-third reported IP to be less of an institutional priority with funding redistributed in 15% of centers [median (IQR) - 25% (- 43, 1)], and resultant staffing changes at 38% of centers. A decrease in IP efforts was reported at 64% of TC. Overall, the majority of respondents reviewed injury data, with the top reported increased mechanisms mainly intentional: Firearm-related (75%), assaults (72%), and abuse (71%). Leading increased unintentional injuries were injuries occurring in the home such as falls (70%), followed by ATV (62%), and cycling (57%). Sites pivoted by presenting (74%) or participating (73%) in IP education virtually, social media posts (61%) and the addition of technology (29%). Top barriers were redeployment of partners (45%) and staff (31%), as well as lack of technology (40%) in the target population. Facilitators were technology at TC (74%), support of trauma program (63%), and having IP funding maintained (55%). CONCLUSIONS: Nearly two-thirds of TC decreased IP efforts during the pandemic due to staffing and funding reductions. The leading reported increased injuries were intentional, indicating that violence prevention is needed, along with support for mental health. While TC successfully pivoted by using technology, access issues in the target population was a barrier resulting in health inequities.

Use of Biostatistical Models to Manage Replicate Error in Concussion Biomarker Research.

Importance: Advancing research on fluid biomarkers associated with sport-related concussion (SRC) highlights the importance of detecting low concentrations using ultrasensitive platforms. However, common statistical practices may overlook replicate errors and specimen exclusion, emphasizing the need to explore robust modeling approaches that consider all available replicate data for comprehensive understanding of sample variation and statistical inferences. Objective: To evaluate the impact of replicate error and different biostatistical modeling approaches on SRC biomarker interpretation. Design, Setting, and Participants: This cross-sectional study within the Surveillance in High Schools to Reduce the Risk of Concussions and Their Consequences study used data from healthy youth athletes (ages 11-18 years) collected from 3 sites across Canada between September 2019 and November 2021. Data were analyzed from November 2022 to February 2023. Exposures: Demographic variables included age, sex, and self-reported history of previous concussion. Main Outcomes and Measures: Outcomes of interest were preinjury plasma glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), neurofilament-light (NFL), total tau (t-tau) and phosphorylated-tau-181 (p-tau-181) assayed in duplicate. Bland-Altman analysis determined the 95% limits of agreement (LOAs) for each biomarker. The impact of replicate error was explored using 3 biostatistical modeling approaches assessing the associations of age, sex, and previous concussion on biomarker concentrations: multilevel regression using all available replicate data, single-level regression using the means of replicate data, and single-level regression with replicate means, excluding specimens demonstrating more than 20% coefficient variation (CV). Results: The sample included 149 healthy youth athletes (78 [52%] male; mean [SD] age, 15.74 [1.41] years; 51 participants [34%] reporting ≥1 previous concussions). Wide 95% LOAs were observed for GFAP (-17.74 to 18.20 pg/mL), UCH-L1 (-13.80 to 14.77 pg/mL), and t-tau (65.27% to 150.03%). GFAP and UCH-L1 were significantly associated with sex in multilevel regression (GFAP: effect size, 15.65%; ß = -0.17; 95% CI, -0.30 to -0.04]; P = .02; UCH-L1: effect size, 17.24%; ß = -0.19; 95% CI, -0.36 to -0.02]; P = .03) and single-level regression using the means of replicate data (GFAP: effect size, 15.56%; ß = -0.17; 95% CI, -0.30 to -0.03]; P = .02; UCH-L1: effect size, 18.02%; ß = -0.20; 95% CI, -0.37 to -0.03]; P = .02); however, there was no association for UCH-L1 after excluding specimens demonstrating more than 20% CV. Excluding specimens demonstrating more than 20% CV resulted in decreased differences associated with sex in GFAP (effect size, 12.29%; ß = -0.14; 95% CI, -0.273 to -0.004]; P = .04) and increased sex differences in UCH-L1 (effect size, 23.59%; ß = -0.27; 95% CI, -0.55 to 0.01]; P = .06), with the widest 95% CIs (ie, least precision) found in UCH-L1. Conclusions and Relevance: In this cross-sectional study of healthy youth athletes, varying levels of agreement between SRC biomarker technical replicates suggested that means of measurements may not optimize precision for population values. Multilevel regression modeling demonstrated how incorporating all available biomarker data could capture replicate variation, avoiding challenges associated with means and percentage of CV exclusion thresholds to produce more representative estimates of association.

Annexin A5 in Patients With Severe COVID-19 Disease: A Single-Center, Randomized, Double-Blind, Placebo-Controlled Feasibility Trial.

OBJECTIVES: To evaluate the study design and feasibility of drug administration and safety in a randomized clinical trial of recombinant human annexin A5 (SY-005), a constitutively expressed protein with anti-inflammatory, antiapoptotic, and anticoagulant properties, in patients with severe coronavirus disease 2019 (COVID-19). DESIGN: Double-blind, randomized clinical trial. SETTING: Two ICUs at an academic medical center. PATIENTS/SUBJECTS: Adults admitted to the ICU with a confirmed diagnosis of COVID-19 and requiring ventilatory or vasopressor support. INTERVENTIONS: SY-005, a recombinant human annexin A5, at 50 or 100 µg/kg IV every 12 hours for 7 days. MEASUREMENTS AND MAIN RESULTS: We enrolled 18 of the 55 eligible patients (33%) between April 21, 2021, and February 3, 2022. We administered 82% (196/238) of the anticipated doses of study medication and 86% (169/196) were given within 1 hour of the scheduled time. There were no drug-related serious adverse events. We captured 100% of the data that would be required for measuring clinical outcomes in a phase 2 or 3 trial. LIMITATIONS: The small sample size was a result of decreasing admissions of patients with COVID-19, which triggered a stopping rule for the trial. CONCLUSIONS: Although enrollment was low, administration of SY-005 to critically ill patients with COVID-19 every 12 hours for up to 7 days was feasible and safe. Further clinical trials of annexin A5 for the treatment of COVID-19 are warranted. Given reduction of severe COVID-19 disease, future studies should explore the safety and effectiveness of SY-005 use in non-COVID-related sepsis.

The utility of syndecan-1 circulating levels as a biomarker in patients with previous or active COVID-19: a systematic review and meta-analysis.

BACKGROUND: With the emergence of coronavirus disease of 2019 (COVID-19), several blood biomarkers have been identified, including the endothelial biomarker syndecan-1, a surface proteoglycan. In the current systematic review and meta-analysis, we aimed to assess the diagnostic and prognostic role of syndecan-1 in COVID-19. METHODS: PubMed, Embase, Scopus, and Web of Science, as international databases, were searched for relevant studies measuring blood syndecan-1 levels in COVID-19 patients, COVID-19 convalescents, and healthy control subjects, in patients with different COVID-19 severities and/or in COVID-19 patients with poor outcomes. Random-effect meta-analysis was performed using STATA to calculate the standardized mean difference (SMD) and 95% confidence interval (CI) for the comparison between COVID-19 patients and healthy control subjects or COVID-19 convalescents and controls. RESULTS: After screening by title/abstract and full text, 17 studies were included in the final review. Meta-analysis of syndecan-1 levels in COVID-19 compared with healthy control subjects revealed that patients with COVID-19 had significantly higher syndecan-1 levels (SMD 1.53, 95% CI 0.66 to 2.41, P < 0.01). In contrast, COVID-19 convalescent patients did not show significant difference with non-convalescents (SMD 0.08, 95% CI -0.63 to 0.78, P = 0.83). Regarding disease severity, two studies reported that more severe forms of the disease were associated with increased syndecan-1 levels. Moreover, patients who died from COVID-19 had higher syndecan-1 levels compared with survivors (SMD 1.22, 95% CI 0.10 to 2.33, P = 0.03). CONCLUSION: Circulating syndecan-1 level can be used as a biomarker of endothelial dysfunction in COVID-19, as it was increased in COVID-19 patients and was higher in more severe instances of the disease. Further larger studies are needed to confirm these findings and further enlighten the role of syndecan-1 in clinical settings.

Tachykinins and the potential causal factors for post-COVID-19 condition.

The most prevalent symptoms of post-COVID-19 condition are pulmonary dysfunction, fatigue and muscle weakness, anxiety, anosmia, dysgeusia, headaches, difficulty in concentrating, sexual dysfunction, and digestive disturbances. Hence, neurological dysfunction and autonomic impairments predominate in post-COVID-19 condition. Tachykinins including the most studied substance P are neuropeptides expressed throughout the nervous and immune systems, and contribute to many physiopathological processes in the nervous, immune, gastrointestinal, respiratory, urogenital, and dermal systems and participate in inflammation, nociception, and cell proliferation. Substance P is a key molecule in neuroimmune crosstalk; immune cells near the peripheral nerve endings can send signals to the brain with cytokines, which highlights the important role of tachykinins in neuroimmune communication. We reviewed the evidence that relates the symptoms of post-COVID-19 condition to the functions of tachykinins and propose a putative pathogenic mechanism. The antagonism of tachykinins receptors can be a potential treatment target.

Plasma proteome of Long-COVID patients indicates HIF-mediated vasculo-proliferative disease with impact on brain and heart function.

AIMS: Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aimed to unveil potential mechanisms, and to inform prognosis and treatment. METHODS: Plasma proteome from Long-COVID outpatients was analyzed in comparison to matched acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of 3072 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity. RESULTS: Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cell redistribution with a dominant resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This potential resetting of cell phenotypes was reflected in prospective vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Several markers (ANGPT1, VEGFA, CCR7, CD56, citrullinated histone 3, elastase) were validated by serological methods in additional patient cohorts. Signaling of transforming growth factor-ß1 with probable connections to elevated EP/p300 suggested vascular inflammation and tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway suggested progression from acute COVID-19 to Long-COVID. The vasculo-proliferative process predicted in Long-COVID might contribute to changes in the organ-specific proteome reflective of neurologic and cardiometabolic dysfunction. CONCLUSIONS: Taken together, our findings point to a vasculo-proliferative process in Long-COVID that is likely initiated either prior hypoxia (localized or systemic) and/or stimulatory factors (i.e., cytokines, chemokines, growth factors, angiotensin, etc). Analyses of the plasma proteome, used as a surrogate for cellular signaling, unveiled potential organ-specific prognostic biomarkers and therapeutic targets.

Organ and cell-specific biomarkers of Long-COVID identified with targeted proteomics and machine learning.

BACKGROUND: Survivors of acute COVID-19 often suffer prolonged, diffuse symptoms post-infection, referred to as "Long-COVID". A lack of Long-COVID biomarkers and pathophysiological mechanisms limits effective diagnosis, treatment and disease surveillance. We performed targeted proteomics and machine learning analyses to identify novel blood biomarkers of Long-COVID. METHODS: A case-control study comparing the expression of 2925 unique blood proteins in Long-COVID outpatients versus COVID-19 inpatients and healthy control subjects. Targeted proteomics was accomplished with proximity extension assays, and machine learning was used to identify the most important proteins for identifying Long-COVID patients. Organ system and cell type expression patterns were identified with Natural Language Processing (NLP) of the UniProt Knowledgebase. RESULTS: Machine learning analysis identified 119 relevant proteins for differentiating Long-COVID outpatients (Bonferonni corrected P < 0.01). Protein combinations were narrowed down to two optimal models, with nine and five proteins each, and with both having excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, F1 = 1.00). NLP expression analysis highlighted the diffuse organ system involvement in Long-COVID, as well as the involved cell types, including leukocytes and platelets, as key components associated with Long-COVID. CONCLUSIONS: Proteomic analysis of plasma from Long-COVID patients identified 119 highly relevant proteins and two optimal models with nine and five proteins, respectively. The identified proteins reflected widespread organ and cell type expression. Optimal protein models, as well as individual proteins, hold the potential for accurate diagnosis of Long-COVID and targeted therapeutics.

Cross-immunity against SARS-COV-2 variants of concern in naturally infected critically ill COVID-19 patients.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

COVID-19 plasma proteome reveals novel temporal and cell-specific signatures for disease severity and high-precision disease management.

Coronavirus disease 2019 (COVID-19) is a systemic inflammatory condition with high mortality that may benefit from personalized medicine and high-precision approaches. COVID-19 patient plasma was analysed with targeted proteomics of 1161 proteins. Patients were monitored from Days 1 to 10 of their intensive care unit (ICU) stay. Age- and gender-matched COVID-19-negative sepsis ICU patients and healthy subjects were examined as controls. Proteomic data were resolved using both cell-specific annotation and deep-analysis for functional enrichment. COVID-19 caused extensive remodelling of the plasma microenvironment associated with a relative immunosuppressive milieu between ICU Days 3-7, and characterized by extensive organ damage. COVID-19 resulted in (1) reduced antigen presentation and B/T-cell function, (2) increased repurposed neutrophils and M1-type macrophages, (3) relatively immature or disrupted endothelia and fibroblasts with a defined secretome, and (4) reactive myeloid lines. Extracellular matrix changes identified in COVID-19 plasma could represent impaired immune cell homing and programmed cell death. The major functional modules disrupted in COVID-19 were exaggerated in patients with fatal outcome. Taken together, these findings provide systems-level insight into the mechanisms of COVID-19 inflammation and identify potential prognostic biomarkers. Therapeutic strategies could be tailored to the immune response of severely ill patients.

Pharmacokinetics of recombinant human annexin A5 (SY-005) in patients with severe COVID-19.

Objective: Annexin A5 is a phosphatidylserine binding protein with anti-inflammatory, anticoagulant and anti-apoptotic properties. Preclinical studies have shown that annexin A5 inhibits pro-inflammatory responses and improves organ function and survival in rodent models of sepsis. This clinical trial aimed to evaluate the pharmacokinetic (PK) properties of the recombinant human annexin A5 (SY-005) in severe COVID-19. Methods: This was a pilot randomized, double-blind, placebo-controlled trial. Severe COVID-19 patients were randomly assigned to receive intravenous 50 µg/kg (low dose, n = 3), 100 µg/kg (high dose, n = 5) of SY-005 or placebo (n = 5) every 12 h for 7 days. Plasma SY-005 levels were assessed using enzyme-linked immunosorbent assay (ELISA) and the PK parameters were determined using non-compartmental analysis. Results: All patients treated with SY-005 had a normal baseline estimated glomerular filtration rate (eGFR, 104-125 mL/min/1.73 m2). Both low and high doses of SY-005 were cleared within 6 h after intravenous administration. Plasma maximum concentrations (Cmax), half-life, clearance and volume distribution of low and high doses of SY-005 were 402.4 and 848.9 ng/mL, 0.92 and 0.96 h, 7.52 and 15.19 L/h, and 9.98 and 20.79 L, respectively. Daily pre-dose circulating annexin A5 levels were not significantly different when SY-005 was administered at the low or the high dose 12-h intervals. There was no significant effect on activated partial thromboplastin time (aPTT) or INR (international normalized ratio of prothrombin time) during 7 days of SY-005 treatment. Conclusion: SY-005 doses of 50 and 100 µg/kg were detectable and subsequently cleared from the plasma in severe COVID-19 patients with normal baseline renal function. There was no significant plasma SY-005 accumulation 6 h after drug administration and coagulation was not altered during 7 days of treatment. Clinical trials Registration: This study was registered with ClinicalTrials.gov (NCT04748757, first posted on 10 February 2021).

Novel plasma protein biomarkers from critically ill sepsis patients.

BACKGROUND: Despite the high morbidity and mortality associated with sepsis, the relationship between the plasma proteome and clinical outcome is poorly understood. In this study, we used targeted plasma proteomics to identify novel biomarkers of sepsis in critically ill patients. METHODS: Blood was obtained from 15 critically ill patients with suspected/confirmed sepsis (Sepsis-3.0 criteria) on intensive care unit (ICU) Day-1 and Day-3, as well as age- and sex-matched 15 healthy control subjects. A total of 1161 plasma proteins were measured with proximal extension assays. Promising sepsis biomarkers were narrowed with machine learning and then correlated with relevant clinical and laboratory variables. RESULTS: The median age for critically ill sepsis patients was 56 (IQR 51-61) years. The median MODS and SOFA values were 7 (IQR 5.0-8.0) and 7 (IQR 5.0-9.0) on ICU Day-1, and 4 (IQR 3.5-7.0) and 6 (IQR 3.5-7.0) on ICU Day-3, respectively. Targeted proteomics, together with feature selection, identified the leading proteins that distinguished sepsis patients from healthy control subjects with ≥ 90% classification accuracy; 25 proteins on ICU Day-1 and 26 proteins on ICU Day-3 (6 proteins overlapped both ICU days; PRTN3, UPAR, GDF8, NTRK3, WFDC2 and CXCL13). Only 7 of the leading proteins changed significantly between ICU Day-1 and Day-3 (IL10, CCL23, TGFα1, ST2, VSIG4, CNTN5, and ITGAV; P < 0.01). Significant correlations were observed between a variety of patient clinical/laboratory variables and the expression of 15 proteins on ICU Day-1 and 14 proteins on ICU Day-3 (P < 0.05). CONCLUSIONS: Targeted proteomics with feature selection identified proteins altered in critically ill sepsis patients relative to healthy control subjects. Correlations between protein expression and clinical/laboratory variables were identified, each providing pathophysiological insight. Our exploratory data provide a rationale for further hypothesis-driven sepsis research.

Initial evaluation of extracorporeal immunomodulatory therapy for the treatment of critically ill COVID-19 infected patients.

Severe COVID-19 infection results in significant immune dysregulation resulting from excessive recruitment and activation of neutrophils. The aim of this study was to confirm feasibility, initial safety and detect signal of efficacy of a non-propriety device delivered using an intermittent extra-corporeal system (LMOD) allowing leucocytes modulation in the setting of Severe COVID-19 infection. Twelve patients were recruited. Inclusion criteria were > 18 years age, confirmed COVID-19, acute respiratory distress syndrome requiring mechanical support and hypotension requiring vasopressor support. Primary end point was vasopressor requirements (expressed as epinephrine dose equivalents) and principle secondary endpoints related to safety, ability to deliver the therapy and markers of inflammation assessed over five days after treatment initiation. LMOD treatment appeared safe, defined by hemodynamic stability and no evidence of white cell number depletion from blood. We demonstrated a significant decrease in vasopressor doses (-37%, p = 0.02) in patients receiving LMOD therapy (despite these patients having to tolerate an additional extracorporeal intermittent therapy). Vasopressor requirements unchanged/increasing in control group (+ 10%, p = 0.48). Although much about the use of this therapy in the setting of severe COVID-19 infection remains to be defined (e.g. optimal dose and duration), this preliminary study supports the further evaluation of this novel extracorporeal approach.

Cohort-Specific Serological Recognition of SARS-CoV-2 Variant RBD Antigens.

OBJECTIVE: Estimating the response of different population cohorts to new SARS-CoV-2 variants is important to customize measures of control. Our goal was to evaluate how antibodies from sera of infected and vaccinated people recognize antigens expressed by different SARS-CoV-2 variants. METHODS: We compared sera from vaccinated donors and four patient/donor cohorts: Sera from critically ill patients collected 2-7 days and more than 10 days after admission to an intensive care unit, a NIBSC/WHO reference panel of SARS-CoV-2 positive individuals, and ambulatory or hospitalized (but not critically ill) positive donors. Samples were tested with an anti-SARS-CoV-2 ELISA kit coated with SARS-CoV-2 RBD recombinant antigens including mutations present in eleven of the most widespread variants. RESULTS: Sera from vaccinated individuals exhibited higher antibody binding (P<0.001) than sera from infected (but not critically ill) individuals when tested against the wild type (WT) and each of 11 variants' receptor binding domain (RBD). Antibodies' binding to the SARS-CoV-2 antigens of at least 6 variants, including Variants of Concern (VOCs), was reduced in comparison to the WT in vaccinated and non-critically ill convalescence individuals. CONCLUSION: Understanding differences between population cohorts in the antibody titers against WT vs variant RBD antigens can help design variant-specific immunoassays for surveillance and evaluation of the epidemiology of new variants.

Elevated vascular transformation blood biomarkers in Long-COVID indicate angiogenesis as a key pathophysiological mechanism.

BACKGROUND: Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated vascular transformation biomarkers in Long-COVID patients. METHODS: A case-control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D. RESULTS: Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05). CONCLUSIONS: Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.