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.

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.

Hereditary cancer genes are highly susceptible to splicing mutations.

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5' and 3' splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing.

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.

Changes in the process of alternative RNA splicing results in soluble B and T lymphocyte attenuator with biological and clinical implications in critical illness.

BACKGROUND: Critically ill patients with sepsis and acute respiratory distress syndrome have severely altered physiology and immune system modifications. RNA splicing is a basic molecular mechanism influenced by physiologic alterations. Immune checkpoint inhibitors, such as B and T Lymphocyte Attenuator (BTLA) have previously been shown to influence outcomes in critical illness. We hypothesize altered physiology in critical illness results in alternative RNA splicing of the immune checkpoint protein, BTLA, resulting in a soluble form with biologic and clinical significance. METHODS: Samples were collected from critically ill humans and mice. Levels soluble BTLA (sBTLA) were measured. Ex vivo experiments assessing for cellular proliferation and cytokine production were done using splenocytes from critically ill mice cultured with sBTLA. Deep RNA sequencing was done to look for alternative splicing of BTLA. sBTLA levels were fitted to models to predict sepsis diagnosis. RESULTS: sBTLA is increased in the blood of critically ill humans and mice and can predict a sepsis diagnosis on hospital day 0 in humans. Alternative RNA splicing results in a premature stop codon that results in the soluble form. sBTLA has a clinically relevant impact as splenocytes from mice with critical illness cultured with soluble BTLA have increased cellular proliferation. CONCLUSION: sBTLA is produced as a result of alternative RNA splicing. This isoform of BTLA has biological significance through changes in cellular proliferation and can predict the diagnosis of sepsis.

Trauma patients who present in a delayed fashion: a unique and challenging population.

BACKGROUND: A proportion of trauma patients present for evaluation in a delayed fashion after injury, likely due to a variety of medical and nonmedical reasons. There has been little investigation into the characteristics and outcomes of trauma patients who present delayed. We hypothesize that trauma patients who present in a delayed fashion are a unique population at risk of increased trauma-related complications. MATERIALS AND METHODS: This was a retrospective review from 2010-2015 at a Level I trauma center. Patients were termed delayed if they presented >24 hours after injury. Patients admitted within 24 hours of their injury were the comparison group. Charts were reviewed for demographics, mechanism, comorbidities, complications and outcomes. A subgroup analysis was done on patients who suffered falls. RESULTS: During the 5-y period, 11,705 patients were admitted. A total of 588 patients (5%) presented >24 h after their injury. Patients in the delayed group were older (65 versus 55 y, P < 0.001) and more likely to have psychiatric comorbidities (33% vs. 24%, P = 0.0001) than the control group. They were also more likely to suffer substance withdrawal (8.9% vs. 4.1%, P < 0.001) but had toxicology testing for drugs and alcohol done at significantly lower rates. Patients that presented delayed after falls were similar in age and injury severity score (ISS) but more likely to suffer substance withdrawal when compared to those with falls that presented within 24 hours. Patients with falls that presented delayed had toxicology testing at significantly lower rates than the comparison group. CONCLUSIONS: Trauma patients that present to the hospital in a delayed fashion have unique characteristics and are more likely to suffer negative outcomes including substance withdrawal. Future goals will include exploring strategies for early intervention, such as automatic withdrawal monitoring and social work referral for all patients who present in a delayed fashion.

Soluble programmed cell death receptor-1 (sPD-1): a potential biomarker with anti-inflammatory properties in human and experimental acute respiratory distress syndrome (ARDS).

BACKGROUND: Acute respiratory distress syndrome (ARDS) remains a common organ dysfunction in the critically ill patient. Mechanisms for its development have focused on immune mediated causes, aspects of our understanding are not complete, and we lack biomarkers. DESIGN, SETTING, AND SUBJECTS: Blood and bronchial alveolar lavage fluid (BAL) from humans (n = 10-13) with ARDS and controls (n = 5-10) as well as a murine model of ARDS (n = 5-6) with controls (n = 6-7) were studied. METHODS: ARDS was induced in mice by hemorrhagic shock (day 1) followed by poly-microbial sepsis (day 2). Samples were then collected on the third day after the animals were euthanized. Ex vivo experiments used splenocytes from animals with ARDS cultured with and without soluble programmed death receptor-1 (sPD-1). RESULTS: Levels of sPD-1 are increased in both the serum (11,429.3 pg/mL(SD 2133.3) vs. 8061.4(SD 4187.8), p = 0.036) and bronchial alveolar lavage (BAL) fluid (6,311.1 pg/mL(SD 3758.0) vs. 90.7 pg/mL(SD 202.8), p = 0.002) of humans with ARDS. Similar results are seen in the serum (9396.1 pg/mL(SD 1546.0) vs. 3464.5 pg/mL(SD 2511.8), p = 0.001) and BAL fluid (2891.7 pg/mL(SD 868.1) vs. 1385.9 pg/mL(SD 927.8), p = 0.012) of mice. sPD-1 levels in murine blood (AUC = 1(1-1), p = 0.006), murine BAL fluid (AUC = 0.905(0.717-1.093), p = 0.015), and human BAL (AUC = 1(1-1), p = 0.001) fluid predicted ARDS. To assess the importance of sPD-1 in ARDS, ex vivo experiments were undertaken. BAL fluid from mice with ARDS dampens the TNF-α production compared to cells cultured with BAL lacking sPD-1 (2.7 pg/mL(SD 3.8) vs. 52.38 pg/mL(SD 25.1), p = 0.002). CONCLUSIONS: This suggests sPD-1 is elevated in critical illness and may represent a potential biomarker for ARDS. In addition, sPD-1 has an anti-inflammatory mechanism in conditions of marked stress and aids in the resolution of severe inflammation. sPD-1 could be used to not only diagnose ARDS, but may be a potential therapy.

Identification of B7-H1 as a novel mediator of the innate immune/proinflammatory response as well as a possible myeloid cell prognostic biomarker in sepsis.

Identifying relevant mediators responsible for the pathogenesis during sepsis may lead to finding novel diagnostic and therapeutic targets. Recent studies indicate programmed cell death receptor (PD)-1 plays a significant role in the development of immune suppression associated with sepsis. In this study, we determine whether B7-H1, the primary ligand of PD-1, contributes to the pathogenesis of sepsis. We report that B7-H1 is upregulated extensively on various immune cells during sepsis and B7-H1 gene deficiency protects mice from the lethality of sepsis. In terms of the histological development of multiple organ damage and inflammatory cytokine levels in circulation or at infectious site, B7-H1-deficient mice showed a remarkable reduction in these indices when compared with wild-type mice. However, B7-H1 gene-deficient mice did not exhibit a lower bacterial burden when compared with wild-type mice, although they recruited more macrophages and neutrophils into infectious site. In addition, we found that, during sepsis, whereas there were no marked differences affecting ex vivo macrophage cytokine productive capacity between PD-1 and B7-H1 gene-deficient mice, preservation of ex vivo macrophage phagocytic function was only seen in septic PD-1 knockout mouse cells. Finally, higher percentage B7-H1(+) neutrophils in peripheral blood correlated not only with higher levels of pro- and anti-inflammatory cytokines/chemokines (CCL2, IL-6, CXCL2, KC, TNF-α, and IL-10), but with lethal outcome as well. Together, these results indicate B7-H1 contributes to septic morbidity in fashion distinct from PD-1 and suggest B7-H1 expression on neutrophils could be used as a biomarker of septic severity.

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.

Empiric antibiotics pending bronchoalveolar lavage data in patients without pneumonia significantly alters the flora, but not the resistance profile, if a subsequent pneumonia develops.

INTRODUCTION: Ventilator-associated pneumonia (VAP) occurs in up to 25% of mechanically ventilated patients, with an associated mortality up to 50%. Early diagnosis and appropriate empiric antibiotic coverage of VAP are crucial. Given the multitude of noninfectious clinical and radiographic anomalies within trauma patients, microbiology from bronchioalveolar lavage (BAL) is often needed. Empiric antibiotics are administered while awaiting BAL culture data. Little is known about the effects of these empiric antibiotics on patients with negative BAL microbiology if a subsequent VAP occurs during the same hospital course. METHODS: This is a retrospective chart review of intubated trauma patients undergoing BAL for suspected pneumonia over a 3-y period at a Level 1 trauma center. All patients with suspected VAP undergoing a BAL receive empiric antibiotics. If microbiology data are negative at 72 h, all antibiotics are stopped; however, if the BAL returns with ≥10(5) colony-forming units per milliliter, the diagnosis of VAP is confirmed. We divided patients into three groups. Group 1 consisted of patients in whom the initial BAL was positive for VAP. Group 2 consisted of patients with an initial negative BAL, who subsequently developed VAP at a later point in the hospital course. Group 3 consisted of patients with negative BAL who did not develop a subsequent VAP. RESULTS: We obtained 499 BAL specimens in 185 patients over the 3-y period. A total of 14 patients with 23 BAL specimens initially negative for VAP subsequently developed VAP later during the same hospital stay. These patients did not have an increase in the hospital length of stay, intensive care unit days, ventilator days, or mortality compared with those who had a positive culture on the first suspicion of VAP. There was a significant increase in the percentage of Enterobacter (21% versus 8%) and Morganella (8% versus 0%) as the causative organism in these 14 patients when the VAP occurred. Furthermore, the profile of the top two organisms in each group changed. Enterobacter (21%) and Pseudomonas (17%) were the principal organisms in the initial BAL-negative group, whereas the two predominant strains in the initial positive BAL group were methicillin-sensitive Staphylococcus aureus (21%) and Haemophilus influenza (11%). Interestingly, methicillin-resistant S. aureus remained the third most common organism in both groups. Empiric antibiotics also did not seem to induce the growth of multidrug-resistant organisms, and there was no increased rate of secondary infections such as Clostridium difficile. CONCLUSIONS: Ventilator-associated pneumonia remains a significant cause of morbidity and mortality in mechanically ventilated trauma patients. The diagnosis and treatment of VAP continue to be challenging. Once clinically suspected, empiric coverage decreases morbidity and mortality. Our data demonstrate that patients who receive empiric coverage exhibit a significantly different microbiologic profile compared with those who had an initial positive BAL culture. Initial empiric antibiotics in BAL-negative patients were not associated with an increase in multidrug-resistant organisms, hospital, or intensive care unit length of stay, ventilator days, and mortality or secondary infections.

B and T lymphocyte attenuator expression on CD4+ T-cells associates with sepsis and subsequent infections in ICU patients.

INTRODUCTION: Sepsis is a deadly inflammatory condition that often leads to an immune suppressed state; however, the events leading to this state remain poorly understood. B and T lymphocyte attenuator (BTLA) is an immune-regulatory receptor shown to effectively inhibit CD4+ T-cell function. Therefore, our objectives were to determine: 1) if lymphocyte BTLA expression was altered in critically ill patients and experimentally induced septic mice, 2) whether augmented CD4+ T-cell BTLA expression was associated with poor septic patient outcomes, and 3) if BTLA expression affected the CD4+ T-cell apoptotic cell loss observed in the lymphoid organs of septic mice. METHODS: Changes in CD4+ lymphocyte BTLA expression were compared with morbid event development in critically ill ICU patients (11 septic and 28 systemic inflammatory response syndrome subjects). Wild type and BTLA gene deficient mice were utilized to evaluate the expression and role of BTLA in septic lymphocyte apoptotic cell loss. RESULTS: The observed septic ICU patients had a significantly higher percentage of peripheral blood BTLA+ CD4+ lymphocytes compared with critically ill non-septic individuals. Moreover, the non-septic patients with CD4+ T-cells that were greater than 80% BTLA+ were more susceptible to developing nosocomial infections. Additionally, in general, critically ill patients with CD4+ T-cells that were greater than 80% BTLA+ had longer hospital stays. Comparatively, circulating CD4+ T-cell and B-cell BTLA expression increased in septic mice, which associated with the increased septic loss of these cells. Finally, the loss of these cells and cellular apoptosis induction in primary and secondary lymphoid organs were reversed in BTLA deficient mice. CONCLUSIONS: An increased BTLA+ CD4+ lymphocyte frequency in the observed critically ill non-septic patients was associated with a subsequent infection; therefore, BTLA may act as a biomarker to help determine nosocomial infection development. Additionally, BTLA expression contributed to primary and secondary lymphoid organ apoptotic cell loss in experimentally septic mice; thus, BTLA-induced apoptotic lymphocyte loss may be a mechanism for increased nosocomial infection risk in critically ill patients. This study had a relatively small human subject cohort; therefore, we feel these findings warrant future studies evaluating the use of BTLA as a critically ill patient nosocomial infection biomarker.

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.

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.

Mechanisms of indirect acute lung injury: a novel role for the coinhibitory receptor, programmed death-1.

OBJECTIVE: To determine the contribution of programmed death receptor (PD)-1 in the morbidity and mortality associated with the development of indirect-acute lung injury. BACKGROUND: The immune cell interaction(s) leading to indirect-acute lung injury are not completely understood. In this respect, we have recently shown that the murine cell surface coinhibitory receptor, PD-1, has a role in septic morbidity/mortality that is mediated in part through the effects on the innate immune arm. However, it is not know if PD-1 has a role in the development of indirect-acute lung injury and how this may be mediated at a cellular level. METHODS: PD-1 -/- mice were used in a murine model of indirect-acute lung injury (hemorrhagic shock followed 24 hours after with cecal ligation and puncture-septic challenge) and compared to wild type controls. Groups were initially compared for survival and subsequently for markers of pulmonary inflammation, influx of lymphocytes and neutrophils, and expression of PD-1 and its ligand-PD-L1. In addition, peripheral blood leukocytes of patients with indirect-acute lung injury were examined to assess changes in cellular PD-1 expression relative to mortality. RESULTS: PD-1 -/- mice showed improved survival compared to wild type controls. In the mouse lung, CD4+, CD11c+, and Gr-1+ cells showed increased PD-1 expression in response to indirect-acute lung injury. However, although the rise in bronchial alveolar lavage fluid protein concentrations, lung IL-6, and lung MCP-1 were similar between PD-1 -/- and wild type animals subjected to indirect acute lung injury, the PD-1 -/- animals that were subjected to shock/septic challenge had reduced CD4:CD8 ratios, TNF-α levels, MPO activity, and Caspase 3 levels in the lung. Comparatively, we observed that humans, who survived their acute lung injury, had significantly lower expression of PD-1 on T cells. CONCLUSIONS: PD-1 expression contributes to mortality after the induction of indirect-acute lung injury and this seems to be associated with modifications in the cellular and cytokine profiles in the lung.

The reflective statement: a new tool to assess resident learning.

PURPOSE: Continued assessment and redesign of the curriculum is essential for optimal surgical education. For the last 3 y, we have asked the residents to reflect on the previous week and describe "the best thing" they learned. We hypothesize that this statement could be used to assess the weaknesses or strengths of our curriculum. METHODS: Starting in 2007, residents filled out surveys approximately 4 times/y at the start of a mandatory conference. They were asked to describe the "best thing" they learned that week, where it was learned, and who taught it. Residents were not asked to classify the item learned by core competency (communication, knowledge, patient care, practice-based learning, professionalism, and systems-based practice). This categorization into core competencies was done as part of our study design. Attending, fellow, resident, or other were used as groups designating who taught each item. Where the item was learned was fit into either clinic, conference, operating room (OR), wards, or self. The impact of postgraduate year (PGY) level on learning was also assessed. χ(2) analysis was used to compare groups. RESULTS: During the study period, 304 surveys were completed and returned by 65 residents. The majority of responses came from PGY 1 residents (134, 43%). Patient care and knowledge were the most common core competencies learned. As PGY level increased, learning of professionalism (P = 0.035) increased. A majority of learning was experiential (wards and OR, P < 0.0125). Self-learning and learning in clinic was a minor component of learning (P < 0.0125). Learning on wards (P < 0.001) decreased as residents progressed and learning from the OR (P = 0.002) had the opposite trend. CONCLUSIONS: Patient care and knowledge are the most frequently cited competencies learned by the residents. Self-learning is not a significant source of learning, and the majority of the learning is experiential. It is not known if this was a sign that there was a lack of self-directed learning or that self-directed learning was not an efficient method of learning. In addition, each PGY level learns differently (teacher and location of learning), perhaps reflecting the different needs and/or structure of each PGY. We believe the reflective statement has been and will be a useful tool to assess our curriculum.

Failure to normalize lymphopenia following trauma is associated with increased mortality, independent of the leukocytosis pattern.

INTRODUCTION: Following trauma and systemic inflammatory response syndrome (SIRS), the typical response is an elevation of the total complete blood count (CBC) and a reduction of the lymphocyte count. This leukocytosis typically returns to normal within 48 hours. The persistence of a leukocytosis following trauma is associated with adverse outcomes. Although lymphocyte anergy and dysfunction following trauma is associated with increased risk for infection and sepsis, there is a paucity of data regarding the impact of a persistence of a low lymphocyte count in trauma patients. METHODS: This is a retrospective review of prospectively collected data from trauma patients collected over the 5 years of September 2003 to September 2008. Patients were included if the injury severity score (ISS) was >or=15, and they survived at least 3 days. Demographic data, mechanism and injury severity score, mortality, and length of stay were collected from the medical record. Laboratory values for the first 4 hospital days were collected. Leukocyte, neutrophil and lymphocyte counts were extracted from the daily complete blood count (CBC). Patients were then grouped based on response (elevation/depression) of each component of the CBC, and their return, or failure thereof, to normal. Proportional hazards regression with time-varying covariates as well as Kaplan-Meier curves were used to predict risk of death, time to death and time to healthy discharge based on fluctuations of the individual components of the CBC. RESULTS: There were 2448 patients admitted over the 5 years included in the analysis. When adjusting for age, gender and ISS the relative risk of death was elevated with a persistent leukocytosis (2.501 (95% CI=1.477-4.235)) or failure to normalize lymphopenia (1.639 (95% CI=10.17-2.643)) within the first 4 days following admission. Similar results were seen when Kaplan-Meier curves were created. Persistent lymphopenia was associated with shortest time to death. Paradoxically in survivors persistent lymphopenia was associated with the shortest time to discharge. CONCLUSIONS: Persistently abnormal CBC responses are associated with a higher mortality following trauma. This is the first report noting that a failure to normalize lymphopenia in severely injured patients is associated with significantly higher mortality.

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.

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.

Impact of socioethnic factors on outcomes following traumatic brain injury.

BACKGROUND: Ethnic minorities and low income families tend to be in poorer health and have worse outcomes for a spectrum of diseases. Health care provider bias has been reported to potentially affect the distribution of care away from poorer communities, minorities, and patients with a history of substance abuse. Trauma is perceived as a disease of the poor and medically underserved. Minorities are overrepresented in low income populations and are also less likely to possess health insurance leading to a potential overlapping effect. Traumatic brain injury (TBI) is a predominant cause of mortality and long-term morbidity, which imposes a considerable social and financial burden. We therefore sought to determine the independent effect on outcome after TBI from race, insurance status, intoxication on presentation, and median income. METHODS: A 5-year retrospective chart review of admitted trauma patients aged 18 years and older to a Level I trauma center. Zip code of residency was a surrogate marker for socioeconomic status, because median income for each zip code is available from the US Census. Charts review included race, insurance status, mechanisms of trauma, and injuries sustained. Outcomes were placement of tracheostomy, hospital length of stay (HLOS), leaving Against Medical Advice (AMA), and discharge to home versus rehabilitation and mortality. RESULTS: A total of 3,101 TBI patients were included in the analyses. Multivariable logistic and proportional hazard regression analyses were undertaken adjusting for age, gender, Injury Severity Score, and mechanism. Rates of tracheostomy placement were unaffected by race, median income, or insurance status. Race and median income did not affect HLOS, but private insurance was associated with shorter HLOS and intoxication was associated with longer HLOS. Neither race nor intoxication affected rates of AMA, but higher income and private insurance was associated with lower rates of AMA. Non-Caucasian race and lack of insurance had significantly lower likelihood of placement in a rehabilitation center. Mortality was unaffected by race, increased in intoxicated patients, was variably affected by median income, and was lowest in patients with private insurance. CONCLUSIONS: An extremely complex interplay exists between socioethnic factors and outcomes after TBI. Few physicians would claim overt discrimination. Tracheostomy, the factor most directed by the surgeon, was unbiased by race, income, or insurance status. The likelihood of placement in a rehabilitation center was significantly impacted by both race and insurance status. Future prospective studies are needed to better address causation.

The development of a urinary tract infection is associated with increased mortality in trauma patients.

BACKGROUND: In October 2008, Medicare and Medicaid stopped paying for care associated with catheter-related urinary tract infections (UTIs). Although most clinicians agree UTIs are detrimental, there are little data to support this belief. METHODS: This is a retrospective review of trauma registry data from a Level I trauma center between 2003 and 2008. Two proportional hazards regressions were used for analyses. The first predicted acquisition of UTI as a function of indwelling urinary catheter use, adjusting for age, diabetes, gender, and injury severity. The second predicted hospital mortality as a function of UTI, covarying for age, gender, chronic obstructive pulmonary disease, congestive heart failure, hypertension, diabetes, pneumonia, and injury severity. RESULTS: After excluding patients who stayed in the hospital <3 days and those with a UTI on arrival, 5,736 patients were included in the study. Of these patients, 680 (11.9%) met criteria for a UTI, with 487 (71.6%) indwelling urinary catheter-related infections. Predictors of UTI included the interaction between age and gender (p = 0.0018), Injury Severity Score (p = 0.0021), and indwelling urinary catheter use (p < 0.001). The development of a UTI predicted the risk of in-hospital death as a patient's age increased (p = 0.002). Similar results were seen when only catheter-associated UTIs are included in the analysis. CONCLUSIONS: Indwelling urinary catheter use is connected to the development of UTIs, and these infections are associated with a greater mortality as the age of a trauma patients increases.