invariant Natural Killer T Cells Modulate the Peritoneal Macrophage Response to Polymicrobial Sepsis.

INTRODUCTION: A dysregulated immune system is a major driver of the mortality and long-term morbidity from sepsis. With respect to macrophages, it has been shown that phenotypic changes are critical to effector function in response to acute infections, including intra-abdominal sepsis. Invariant natural killer T cells (iNKT cells) have emerged as potential central regulators of the immune response to a variety of infectious insults. Specifically, various iNKT cell:macrophage interactions have been noted across a spectrum of diseases, including acute events such as sepsis. However, the potential for iNKT cells to affect peritoneal macrophages during an abdominal septic event is as yet unknown. METHODS: Cecal ligation and puncture (CLP) was performed in both wild type (WT) and invariant natural killer T cell knockout (iNKT-/-) mice. 24 h following CLP or sham operation, peritoneal macrophages were collected for analysis. Analysis of macrophage phenotype and function was undertaken to include analysis of bactericidal activity and cytokine or superoxide production. RESULTS: Within iNKT-/- mice, a greater degree of intraperitoneal macrophages in response to the sepsis was noted. Compared to WT mice, within iNKT-/- mice, CLP did induce an increase in CD86+ and CD206+, but no difference in CD11b+. Unlike WT mice, intra-abdominal sepsis within iNKT-/- mice induced an increase in Ly6C-int (5.2% versus 14.9%; P < 0.05) and a decrease in Ly6C-high on peritoneal macrophages. Unlike phagocytosis, iNKT cells did not affect macrophage bactericidal activity. Although iNKT cells did not affect interleukin-6 production, iNKT cells did affect IL-10 production and both nitrite and superoxide production from peritoneal macrophages. CONCLUSIONS: The observations indicate that iNKT cells affect specific phenotypic and functional aspects of peritoneal macrophages during polymicrobial sepsis. Given that pharmacologic agents that affect iNKT cell functioning are currently in clinical trial, these findings may have the potential for translation to critically ill surgical patients with abdominal sepsis.

The unusual gene architecture of polyubiquitin is created by dual-specific splice sites.

BACKGROUND: The removal of introns occurs through the splicing of a 5' splice site (5'ss) with a 3' splice site (3'ss). These two elements are recognized by distinct components of the spliceosome. However, introns in higher eukaryotes contain many matches to the 5' and 3' splice-site motifs that are presumed not to be used. RESULTS: Here, we find that many of these sites can be used. We also find occurrences of the AGGT motif that can function as either a 5'ss or a 3'ss-previously referred to as dual-specific splice sites (DSSs)-within introns. Analysis of the Sequence Read Archive reveals a 3.1-fold enrichment of DSSs relative to expectation, implying synergy between the ability to function as a 5'ss and 3'ss. Despite this suggested mechanistic advantage, DSSs are 2.7- and 4.7-fold underrepresented in annotated 5' and 3' splice sites. A curious exception is the polyubiquitin gene UBC, which contains a tandem array of DSSs that precisely delimit the boundary of each ubiquitin monomer. The resulting isoforms splice stochastically to include a variable number of ubiquitin monomers. We found no evidence of tissue-specific or feedback regulation but note the 8.4-fold enrichment of DSS-spliced introns in tandem repeat genes suggests a driving role in the evolution of genes like UBC. CONCLUSIONS: We find an excess of unannotated splice sites and the utilization of DSSs in tandem repeats supports the role of splicing in gene evolution. These findings enhance our understanding of the diverse and complex nature of the splicing process.

Transcriptional profiles of pulmonary artery endothelial cells in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This "cell biopsy" method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.

Application of single cell multiomics points to changes in chromatin accessibility near calcitonin receptor like receptor and a possible role for adrenomedullin in the post-shock lung.

Introduction: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a commonly occurring sequelae of traumatic injury resulting from indirect insults like hypovolemic shock and/or extrapulmonary sepsis. The high lethality rate associated with these pathologies outlines the importance of clarifying the "priming" effects seen in the post-shock lung microenvironment, which are understood to bring about a dysregulated or overt immune response when triggered by a secondary systemic infectious/septic challenge culminating in ALI. In this pilot project, we test the hypothesis that application of a single cell multiomics approach can elucidate novel phenotype specific pathways potentially contributing to shock-induced ALI/ARDS. Methods: Hypovolemic shock was induced in C57BL/6 (wild-type), PD-1, PD-L1, or VISTA gene deficient male mice, 8-12 weeks old. Wild-type sham surgeries function as negative controls. A total of 24-h post-shock rodents were sacrificed, their lungs harvested and sectioned, with pools prepared from 2 mice per background, and flash frozen on liquid nitrogen. N = 2 biological replicates (representing 4 mice total) were achieved for all treatment groups across genetic backgrounds. Samples were received by the Boas Center for Genomics and Human Genetics, where single cell multiomics libraries were prepared for RNA/ATAC sequencing. The analysis pipeline Cell Ranger ARC was implemented to attain feature linkage assessments across genes of interest. Results: Sham (pre-shock) results suggest high chromatin accessibility around calcitonin receptor like receptor (CALCRL) across cellular phenotypes with 17 and 18 feature links, exhibiting positive correlation with gene expression between biological replicates. Similarity between both sample chromatin profiles/linkage arcs is evident. Post-shock wild-type accessibility is starkly reduced across replicates where the number of feature links drops to 1 and 3, again presenting similar replicate profiles. Samples from shocked gene deficient backgrounds displayed high accessibility and similar profiles to the pre-shock lung microenvironment. Conclusion: High pre-shock availability of DNA segments and their positive correlation with CALCRL gene expression suggests an apparent regulatory capacity on transcription. Post-shock gene deficient chromatin profiles presented similar results to that of pre-shock wild-type samples, suggesting an influence on CALCRL accessibility. Key changes illustrated in the pre-ALI context of shock may allow for additional resolution of "priming" and "cellular pre-activation/pre-disposition" processes within the lung microenvironment.

Deep RNA sequencing of intensive care unit patients with COVID-19.

COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (> 100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies.

Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations.

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments. Clinical data were prospectively collected at multiple time points during ICU admission, and amino acid sequences for the NAb CDR3 segments were identified. Patients who survived severe COVID-19 had significantly more of a Class 3 antibody (C135) to SARS-CoV-2 compared to non-survivors (15059.4 vs. 1412.7, p = 0.016). In addition to highlighting the utility of RNA sequencing in revealing unique NAb profiles in COVID-19 patients with different outcomes, we provided a physical basis for our findings via atomistic modeling combined with molecular dynamics simulations. We established the interactions of the Class 3 NAb C135 with the SARS-CoV-2 spike protein, proposing a mechanistic basis for inhibition via multiple conformations that can effectively prevent ACE2 from binding to the spike protein, despite C135 not directly blocking the ACE2 binding motif. Overall, we demonstrate that deep RNA sequencing combined with structural modeling offers the new potential to identify and understand novel therapeutic(s) NAbs in individuals lacking certain immune responses due to their poor endogenous production. Our results suggest a possible window of opportunity for administration of such NAbs when their full sequence becomes available. A method involving rapid deep RNA sequencing of patients infected with SARS-CoV-2 or its variants at the earliest infection time could help to develop personalized treatments using the identified specific NAbs.

Efficient Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from Exhaled Breath.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is transmitted through airborne particles in exhaled breath, causing severe respiratory disease, coronavirus disease-2019 (COVID-19), in some patients. Samples for SARS-CoV-2 testing are typically collected by nasopharyngeal swab, with the virus detected by PCR; however, patients can test positive for 3 months after infection. Without the capacity to assay SARS-CoV-2 in breath, it is not possible to understand the risk for transmission from infected individuals. To detect virus in breath, the Bubbler-a breathalyzer that reverse-transcribes RNA from SARS-CoV-2 particles into a sample-specific barcoded cDNA-was developed. In a study of 70 hospitalized patients, the Bubbler was both more predictive of lower respiratory tract involvement (abnormal chest X-ray) and less invasive than alternatives. Samples tested using the Bubbler were threefold more enriched for SARS-CoV-2 RNA than were samples from tongue swabs, implying that virus particles were being directly sampled. The barcode-enabled Bubbler was used for simultaneous diagnosis in large batches of pooled samples at a lower limit of detection of 334 genomic copies per sample. Diagnosis by sequencing can provide additional information, such as viral load and strain identity. The Bubbler was configured to sample nucleic acids in water droplets circulating in air, demonstrating its potential in environmental monitoring and the protective effect of adequate ventilation.

Emerging therapeutic targets for sepsis.

Introduction: Sepsis is characterized by a dysregulated host response to infection. Sepsis-associated morbidity/mortality demands concerted research efforts toward therapeutic interventions which are reliable, broadly effective, and etiologically based. More intensive and extensive investigations on alterations in cellular signaling pathways, gene targeting as a means of modifying the characteristic hyper and/or hypo-immune responses, prevention through optimization of the microbiome, and the molecular pathways underlying the septic immune response could improve outcomes.] Areas covered: The authors discuss key experimental mammalian models and clinical trials. They provide an evaluation of evolving therapeutics in sepsis and how they have built upon past and current treatments. Relevant literature was derived from a PubMed search spanning 1987-2020.Expert opinion: Given the complex nature of sepsis and the elicited immune response, it is not surprising that a single cure-all therapeutic intervention, which is capable of effectively and reliably improving patient outcomes has failed to emerge. Innovative approaches seek to address not only the disease process but modify underlying patient factors. A true improvement in sepsis-associated morbidity/mortality will require a combination of unique therapeutic modalities.

Deep RNA Sequencing of Intensive Care Unit Patients with COVID-19.

PURPOSE: COVID-19 has impacted millions of patients across the world. Molecular testing occurring now identifies the presence of the virus at the sampling site: nasopharynx, nares, or oral cavity. RNA sequencing has the potential to establish both the presence of the virus and define the host's response in COVID-19. METHODS: Single center, prospective study of patients with COVID-19 admitted to the intensive care unit where deep RNA sequencing (>100 million reads) of peripheral blood with computational biology analysis was done. All patients had positive SARS-CoV-2 PCR. Clinical data was prospectively collected. RESULTS: We enrolled fifteen patients at a single hospital. Patients were critically ill with a mortality of 47% and 67% were on a ventilator. All the patients had the SARS-CoV-2 RNA identified in the blood in addition to RNA from other viruses, bacteria, and archaea. The expression of many immune modulating genes, including PD-L1 and PD-L2, were significantly different in patients who died from COVID-19. Some proteins were influenced by alternative transcription and splicing events, as seen in HLA-C, HLA-E, NRP1 and NRP2. Entropy calculated from alternative RNA splicing and transcription start/end predicted mortality in these patients. CONCLUSIONS: Current upper respiratory tract testing for COVID-19 only determines if the virus is present. Deep RNA sequencing with appropriate computational biology may provide important prognostic information and point to therapeutic foci to be precisely targeted in future studies. TAKE HOME MESSAGE: Deep RNA sequencing provides a novel diagnostic tool for critically ill patients. Among ICU patients with COVID-19, RNA sequencings can identify gene expression, pathogens (including SARS-CoV-2), and can predict mortality. TWEET: Deep RNA sequencing is a novel technology that can assist in the care of critically ill COVID-19 patients & can be applied to other disease.

Unjustified Administration in Liberal Use of Tranexamic Acid in Trauma Resuscitation.

BACKGROUND: Early administration of tranexamic acid (TXA) has been widely implemented for the treatment of presumed hyperfibrinolysis in hemorrhagic shock. We aimed to characterize the liberal use of TXA and whether unjustified administration was associated with increased venous thrombotic events (VTEs). METHODS: We identified injured patients who received TXA between January 2016 and January 2018 by querying our Level 1 trauma center's registry. We retrospectively reviewed medical records and radiologic images to classify whether patients had a hemorrhagic injury that would have benefited from TXA (justified) or not (unjustified). RESULTS: Ninety-five patients received TXA for traumatic injuries, 42.1% were given by emergency medical services. TXA was considered unjustified in 35.8% of the patients retrospectively and in 52% of the patients when given by emergency medical services. Compared with unjustified administration, patients in the justified group were younger (47.6 versus 58.4; P = 0.02), more hypotensive in the field (systolic blood pressure: 107 ± 31 versus 137 ± 32 mm Hg; P < 0.001) and in the emergency department (systolic blood pressure: 97 ± 27 versus 128 ± 27; P < 0.001), and more tachycardic in emergency department (heart rate: 99 ± 29 versus 88 ± 19; P = 0.04). The justified group also had higher injury severity score (median 24 versus 11; P < 0.001), was transfused more often (81.7% versus 20.6%; P < 0.001), and had higher in-hospital mortality (39.3% versus 2.9%; P < 0.001), but there was no difference in the rate of VTE (8.2% versus 5.9%). CONCLUSIONS: Our results highlight a high rate of unjustified administration, especially in the prehospital setting. Hypotension and tachycardia were indications of correct use. Although we did not observe a difference in VTE rates between the groups, though, our study was underpowered to detect a difference. Cautious implementation of TXA in resuscitation protocols is encouraged in the meantime. Nonetheless, adverse events associated with unjustified TXA administration should be further evaluated.

Herpes Virus Entry Mediator (HVEM): A Novel Potential Mediator of Trauma-Induced Immunosuppression.

BACKGROUND: Herpes virus entry mediator (HVEM) is a coinhibitory molecule which can both stimulate and inhibit host immune responses. Altered expression of HVEM and its ligands is associated with increased nosocomial infections in septic patients. We hypothesize critically ill trauma patients will display increased lymphocyte HVEM expression and that such alteration is predictive of infectious events. MATERIALS AND METHODS: Trauma patients prospectively enrolled from the ICU were compared with healthy controls. Leukocytes were isolated from whole blood, stained for CD3 (lymphocytes) and HVEM, and evaluated by flow cytometry. Charts were reviewed for injuries sustained, APACHE II score, hospital course, and secondary infections. RESULTS: Trauma patients (n = 31) were older (46.7 ± 2.4 versus 36.8 ± 2.1 y; P = 0.03) than healthy controls (n = 10), but matched for male sex (74% versus 60%; P = 0.4). Trauma patients had higher presenting WBC (13.9 ± 1.3 versus 5.6 ± 0.5 × 106/mL; P = 0.002), lower percentage of CD3+ lymphocytes (7.5% ± 0.8 versus 22.5% ± 0.9; P < 0.001), but significantly greater expression of HVEM+/CD3+ lymphocytes (89.6% ± 1.46 versus 67.3% ± 1.7; P < 0.001). Among trauma patients, secondary infection during the hospitalization was associated with higher APACHE II scores (20.6 ± 1.6 versus 13.6 ± 1.4; P = 0.03) and markedly lower CD3+ lymphocyte HVEM expression (75% ± 2.6 versus 93% ± 0.7; P < 0.01). CONCLUSIONS: HVEM expression on CD3+ cells increases after trauma. Patients developing secondary infections have less circulating HVEM+CD3+. This implies HVEM signaling in lymphocytes plays a role in maintaining host defense to infection in after trauma. HVEM expression may represent a marker of infectious risk as well as a potential therapeutic target, modulating immune responses to trauma.

Check Point Inhibitors and Their Role in Immunosuppression in Sepsis.

Checkpoint regulators are a group of membrane-bound receptors or ligands expressed on immune cells to regulate the immune cell response to antigen presentation and other immune stimuli, such as cytokines, chemokines, and complement. In the context of profound immune activation, such as sepsis, the immune system can be rendered anergic by these receptors to prevent excessive inflammation and tissue damage. If this septic immunosuppression is prolonged, the host is unable to mount the appropriate immune response to a secondary insult or infection. This article describes the manner in which major regulators in the B7-CD28 family and their ligands mediate immunosuppression in sepsis.

Dismal outcomes following damage control laparotomy in injured older adults, a cohort study.

BACKGROUND: The population of older adults is rapidly growing and more older patients are presenting with abdominal trauma. Outcomes have not been well defined for patients that require a damage control approach(DCL). METHODS: This was a retrospective study at a level one trauma center of patients age 65 years and older with abdominal trauma that required DCL. Outcomes reviewed included mortality, length of stay, discharge disposition. Presenting vital signs and laboratories were reviewed to identify predictors of mortality. RESULTS: 31 older patients(mean age 75.2 years) underwent DCL. Twenty-four of 31(77.4%) older patients died. Seven of 7 older DCL survivors were discharged to a rehabilitation center or nursing home. In comparisons of older DCL nonsurvivors and survivors there were not differences in presenting HR(90 versus 96; p = 0.56) or SBP in the emergency room(107 versus 116; p = 0.51). No differences in initial lactate or change in lactate concentration were found between nonsurvivors and survivors. Fifteen of 24 nonsurvivors died from multisystem organ failure. CONCLUSIONS/IMPLICATIONS: The mortality rate of older patients that require damage control approach for is extremely high. Presenting vital signs and laboratory markers may not be useful in older patients to predict mortality.