Angiogenesis-associated pathways play critical roles in neonatal sepsis outcomes.

Neonatal sepsis is a major cause of childhood mortality. Limited diagnostic tools and mechanistic insights have hampered our abilities to develop prophylactic or therapeutic interventions. Biomarkers in human neonatal sepsis have been repeatedly identified as associated with dysregulation of angiopoietin signaling and altered arachidonic acid metabolism. We here provide the mechanistic evidence in support of the relevance for these observations. Angiopoetin-1 (Ang-1), which promotes vascular integrity, was decreased in blood plasma of human and murine septic newborns. In preclinical models, administration of Ang-1 provided prophylactic protection from septic death. Arachidonic acid metabolism appears to be functionally connected to Ang-1 via reactive oxygen species (ROS) with a direct role of nitric oxide (NO). Strengthening this intersection via oral administration of arachidonic acid and/or the NO donor L-arginine provided prophylactic as well as therapeutic protection from septic death while also increasing plasma Ang-1 levels among septic newborns. Our data highlight that targeting angiogenesis-associated pathways with interventions that increase Ang-1 activity directly or indirectly through ROS/eNOS provide promising avenues to prevent and/or treat severe neonatal sepsis.

Post-COVID symptoms are associated with endotypes reflecting poor inflammatory and hemostatic modulation.

Introduction: Persistent symptoms after COVID-19 infection ("long COVID") negatively affects almost half of COVID-19 survivors. Despite its prevalence, its pathophysiology is poorly understood, with multiple host systems likely affected. Here, we followed patients from hospital to discharge and used a systems-biology approach to identify mechanisms of long COVID. Methods: RNA-seq was performed on whole blood collected early in hospital and 4-12 weeks after discharge from 24 adult COVID-19 patients (10 reported post-COVID symptoms after discharge). Differential gene expression analysis, pathway enrichment, and machine learning methods were used to identify underlying mechanisms for post-COVID symptom development. Results: Compared to patients with post-COVID symptoms, patients without post-COVID symptoms had larger temporal gene expression changes associated with downregulation of inflammatory and coagulation genes over time. Patients could also be separated into three patient endotypes with differing mechanistic trajectories, which was validated in another published patient cohort. The "Resolved" endotype (lowest rate of post-COVID symptoms) had robust inflammatory and hemostatic responses in hospital that resolved after discharge. Conversely, the inflammatory/hemostatic responses of "Suppressive" and "Unresolved" endotypes (higher rates of patients with post-COVID symptoms) were persistently dampened and activated, respectively. These endotypes were accurately defined by specific blood gene expression signatures (6-7 genes) for potential clinical stratification. Discussion: This study allowed analysis of long COVID whole blood transcriptomics trajectories while accounting for the issue of patient heterogeneity. Two of the three identified and externally validated endotypes ("Unresolved" and "Suppressive") were associated with higher rates of post-COVID symptoms and either persistently activated or suppressed inflammation and coagulation processes. Gene biomarkers in blood could potentially be used clinically to stratify patients into different endotypes, paving the way for personalized long COVID treatment.

Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness.

The COVID-19 pandemic has created an urgency to study the host gene response that leads to variable clinical presentations of the disease, particularly the critical illness response. miRNAs have been implicated in the mechanism of host immune dysregulation and thus hold potential as biomarkers and/or therapeutic agents with clinical application. Hence, further analyses of their altered expression in COVID-19 is warranted. An important basis for this is identifying appropriate reference genes for high quality expression analysis studies. In the current report, NanoString technology was used to study the expression of 798 miRNAs in the peripheral blood of 24 critically ill patients, 12 had COVID-19 and 12 were COVID-19 negative. A list of potentially stable candidate reference genes was generated that included ten miRNAs. The top six were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a total of 41 patients so as to apply standard computational algorithms for validating reference genes, namely geNorm, NormFinder, BestKeeper and RefFinder. There was general agreement among all four algorithms in the ranking of four stable miRNAs: miR-186-5p, miR-148b-3p, miR-194-5p and miR-448. A detailed analysis of their output rankings led to the conclusion that miR-186-5p and miR-148b-3p are appropriate reference genes for miRNA expression studies using PaxGene tubes in the peripheral blood of patients critically ill with COVID-19 disease.

Predicting severity in COVID-19 disease using sepsis blood gene expression signatures.

Severely-afflicted COVID-19 patients can exhibit disease manifestations representative of sepsis, including acute respiratory distress syndrome and multiple organ failure. We hypothesized that diagnostic tools used in managing all-cause sepsis, such as clinical criteria, biomarkers, and gene expression signatures, should extend to COVID-19 patients. Here we analyzed the whole blood transcriptome of 124 early (1-5 days post-hospital admission) and late (6-20 days post-admission) sampled patients with confirmed COVID-19 infections from hospitals in Quebec, Canada. Mechanisms associated with COVID-19 severity were identified between severity groups (ranging from mild disease to the requirement for mechanical ventilation and mortality), and established sepsis signatures were assessed for dysregulation. Specifically, gene expression signatures representing pathophysiological events, namely cellular reprogramming, organ dysfunction, and mortality, were significantly enriched and predictive of severity and lethality in COVID-19 patients. Mechanistic endotypes reflective of distinct sepsis aetiologies and therapeutic opportunities were also identified in subsets of patients, enabling prediction of potentially-effective repurposed drugs. The expression of sepsis gene expression signatures in severely-afflicted COVID-19 patients indicates that these patients should be classified as having severe sepsis. Accordingly, in severe COVID-19 patients, these signatures should be strongly considered for the mechanistic characterization, diagnosis, and guidance of treatment using repurposed drugs.

Identifying clinical subtypes in sepsis-survivors with different one-year outcomes: a secondary latent class analysis of the FROG-ICU cohort.

BACKGROUND: Late mortality risk in sepsis-survivors persists for years with high readmission rates and low quality of life. The present study seeks to link the clinical sepsis-survivors heterogeneity with distinct biological profiles at ICU discharge and late adverse events using an unsupervised analysis. METHODS: In the original FROG-ICU prospective, observational, multicenter study, intensive care unit (ICU) patients with sepsis on admission (Sepsis-3) were identified (N = 655). Among them, 467 were discharged alive from the ICU and included in the current study. Latent class analysis was applied to identify distinct sepsis-survivors clinical classes using readily available data at ICU discharge. The primary endpoint was one-year mortality after ICU discharge. RESULTS: At ICU discharge, two distinct subtypes were identified (A and B) using 15 readily available clinical and biological variables. Patients assigned to subtype B (48% of the studied population) had more impaired cardiovascular and kidney functions, hematological disorders and inflammation at ICU discharge than subtype A. Sepsis-survivors in subtype B had significantly higher one-year mortality compared to subtype A (respectively, 34% vs 16%, p < 0.001). When adjusted for standard long-term risk factors (e.g., age, comorbidities, severity of illness, renal function and duration of ICU stay), subtype B was independently associated with increased one-year mortality (adjusted hazard ratio (HR) = 1.74 (95% CI 1.16-2.60); p = 0.006). CONCLUSIONS: A subtype with sustained organ failure and inflammation at ICU discharge can be identified from routine clinical and laboratory data and is independently associated with poor long-term outcome in sepsis-survivors. Trial registration NCT01367093; https://clinicaltrials.gov/ct2/show/NCT01367093 .

The Impact of Migration on the Gut Metagenome of South Asian Canadians.

South Asian (SA) Canadian immigrants have a higher risk of developing certain immune-mediated inflammatory diseases compared to non-migrant SAs. We sought to investigate the effect of migration on the gut metagenome and to identify microbiological associations between migration and conditions that may influence the development of immune-mediated inflammatory diseases. Metagenomic analysis of 58 first-generation (GEN1) SA immigrants and 38 unrelated Canadian born children-of-immigrants (GEN2) determined that the time lived in Canada was associated with continued changes in gut microbial communities. Migration of GEN1 to Canada early in life results in a gut community with similarities to GEN2 SA Canadians and non-SA North Americans. Conversely, GEN1 immigrants who arrived recently to Canada exhibited pronounced differences from GEN2, while displaying microbial similarities to a non-migrating SA cohort. Multivariate analysis identified that community composition was primarily influenced by high abundance taxa. Prevotella copri dominated in GEN1 and non-migrant SAs. Clostridia and functionally related Bacteroidia spp. replaced P. copri dominance over generations in Canada. Mutually exclusive Dialister species occurred at differing relative abundances over time and generations in Canada. This shift in species composition is accompanied by a change in genes associated with carbohydrate utilization and short-chain fatty acid production. Total energy derived from carbohydrates compared to protein consumption was significantly higher for GEN1 recent immigrants, which may influence the functional requirements of the gut community. This study demonstrates the associations between migration and the gut microbiome, which may be further associated with the altered risk of immune-mediated inflammatory diseases observed for SA Canadians.

Comparative functional genomic screens of three yeast deletion collections reveal unexpected effects of genotype in response to diverse stress.

The Yeast Knockout (YKO) collection has provided a wealth of functional annotations from genome-wide screens. An unintended consequence is that 76% of gene annotations derive from one genotype. The nutritional auxotrophies in the YKO, in particular, have phenotypic consequences. To address this issue, 'prototrophic' versions of the YKO collection have been constructed, either by introducing a plasmid carrying wild-type copies of the auxotrophic markers (Plasmid-Borne, PBprot) or by backcrossing (Backcrossed, BCprot) to a wild-type strain. To systematically assess the impact of the auxotrophies, genome-wide fitness profiles of prototrophic and auxotrophic collections were compared across diverse drug and environmental conditions in 250 experiments. Our quantitative profiles uncovered broad impacts of genotype on phenotype for three deletion collections, and revealed genotypic and strain-construction-specific phenotypes. The PBprot collection exhibited fitness defects associated with plasmid maintenance, while BCprot fitness profiles were compromised due to strain loss from nutrient selection steps during strain construction. The repaired prototrophic versions of the YKO collection did not restore wild-type behaviour nor did they clarify gaps in gene annotation resulting from the auxotrophic background. To remove marker bias and expand the experimental scope of deletion libraries, construction of a bona fide prototrophic collection from a wild-type strain will be required.

Standardizing leucocyte PNH clone detection: an international study.

BACKGROUND: Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol-deficient structures on red blood cells and white blood cells in paroxysmal nocturnal hemoglobinuria (PNH) were recently published. METHODS: UK NEQAS LI issued three stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific Standard Operating Procedures (SOPs) and pretitered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH External Quality Assessment (EQA) programs. RESULTS: Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their "in-house" methods, though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an interquartile range of 1.7% and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. CONCLUSIONS: The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen among the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories.

Standardizing Leucocyte PNH clone detection: An international study.

Background: Consensus and Practical Guidelines for robust high-sensitivity detection of glycophosphatidylinostitol (GPI)-deficient structures on Red Blood Cells (RBCs) and White Blood Cells (WBCs) in Paroxysmal Nocturnal Hemoglobinuria (PNH) were recently published. Methods: UK NEQAS LI issued 3 stabilized samples manufactured to contain no PNH cells (normal), approximately 0.1% and 8% PNH leucocyte populations, together with instrument-specific SOPs and pre-titered antibody cocktails to 19 international laboratories experienced in PNH testing. Samples were tested using both standardized protocol/reagents and in-house protocols. Additionally, samples were issued to all participants in the full PNH EQA programmes. Results: Expert laboratory results showed no difference in PNH clone detection rates when using standardized and their 'in-house' methods though lower variation around the median was found for the standardized approach compared to in-house methods. Neutrophil analysis of the sample containing an 8% PNH population, for example, showed an interquartile range of 0.48% with the standardized approach compared with 1.29% for in-house methods. Results from the full EQA group showed the greatest variation with an inter-quartile range of 1.70 and this was demonstrated to be significantly different (P<0.001) to the standardized cohort. Conclusions: The results not only demonstrate that stabilized whole PNH blood samples are suitable for use with currently recommended high-sensitivity reagent cocktails/protocols but also highlight the importance of using carefully selected conjugates alongside the standardized protocols. While much more variation was seen amongst the full UK NEQAS LI EQA group, the standardized approach lead to reduced variation around the median even for the experienced laboratories. © 2014 Clinical Cytometry Society.

Use of a FLAER-based WBC assay in the primary screening of PNH clones.

Diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) with flow cytometry traditionally involves the analysis of CD55 and CD59 on RBCs and neutrophils. However, the ability to accurately detect PNH RBCs is compromised by prior hemolysis and/or transfused RBCs. Patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS) can also produce PNH clones. We recently described a multiparameter fluorescent aerolysin (FLAER)-based flow assay using CD45, CD33, and CD14 that accurately identified PNH monocyte and neutrophil clones in PNH, AA, and MDS. Here, we compared the efficiency of this WBC assay with a CD59-based assay on RBCs during a 3-year period. PNH clones were detected with the FLAER assay in 63 (11.8%) of 536 samples tested, whereas PNH RBCs were detected in only 33 (6.2%), and always with a smaller clone size. The FLAER assay on WBCs is a more sensitive and robust primary screening assay for detecting PNH clones in clinical samples.

Comparison of two single-platform ISHAGE-based CD34 enumeration protocols on BD FACSCalibur and FACSCanto flow cytometers.

BACKGROUND AIMS: Enumeration of viable CD34(+) cells provides critical information for the bone marrow (BM) transplant physician. The single-platform ISHAGE protocol is the most reliable method currently available to quantitate accurately this important subset of cells. Previous studies have shown that 5 CD34(+) cells/microL blood predicts the collection of at least 0.5x10(6) CD34(+) cells/kg patient weight. From the apheresis product, infusion of 2.5x10(6) viable CD34(+) cells (measured pre-cryopreservation)/kg patient weight will reliably permit engraftment of the hematopoietic system (as measured by the time to 20000 platelets/microL) by day 12-14 post-infusion. METHODS: We compared the CD34(+) cell numbers derived from Flow Count-based Stem-Kit; (Beckman Coulter) and Trucount tube-based stem cell enumeration (SCE) kit (BD Biosciences) ISHAGE templates on BD FACSCalibur and BD FACSCanto cytometers on 12 granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) and 10 peripheral blood stem cell (PBSC) samples. RESULTS: Comparison of results showed that there was no statistical difference between samples run with Stem-Kit on the FACSCalibur versus SCE kit-based assays on either the FACSCalibur or FACSCanto. Mean results for the Stem-Kit/Calibur combination were 137, for SCE kit/Calibur 140 and for SCE kit/Canto 137 cells/microL. Pair-wise comparison of data based on rank order showed no statistically significant difference and all correlation coefficients had an R(2)>0.98. CONCLUSIONS: The two kits generated very similar data on a range of fresh samples regardless of instrument platform. These results confirm and extend the utility of the single-platform ISHAGE protocols with a variety of reagent kits and instrument platforms.