Identification of unique microRNA expression patterns in bone marrow hematopoietic stem and progenitor cells after hemorrhagic shock and multiple injuries in young and old adult mice.

BACKGROUND: After severe trauma, the older host experiences more dysfunctional hematopoiesis of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs), and dysfunctional differentiation of circulating myeloid cells into effective innate immune cells. Our main objective was to compare BM HSPC microRNA (miR) responses of old and young mice in a clinically relevant model of severe trauma and shock. METHODS: C57BL/6 adult male mice aged 8 to 12 weeks (young) and 18 to 24 months (old) underwent multiple injuries and hemorrhagic shock (polytrauma [PT]) that engenders the equivalent of major trauma (Injury Severity Score, >15). Pseudomonas pneumonia (PNA) was induced in some young and old adult mice 24 hours after PT. MicroRNA expression patterns were determined from lineage-negative enriched BM HSPCs isolated from PT and PT-PNA mice at 24 and 48 hours postinjury, respectively. Genome-wide expression and pathway analyses were also performed on bronchoalveolar lavage (BAL) leukocytes from both mouse cohorts. RESULTS: MicroRNA expression significantly differed among all experimental conditions (p < 0.05), except for old-naive versus old-injured (PT or PT-PNA) mice, suggesting an inability of old mice to mount a robust early miR response to severe shock and injury. In addition, young adult mice had significantly more leukocytes obtained from their BAL, and there were greater numbers of polymorphonuclear cells compared with old mice (59.8% vs. 2.2%, p = 0.0069). Despite increased gene expression changes, BAL leukocytes from old mice demonstrated a more dysfunctional transcriptomic response to PT-PNA than young adult murine BAL leukocytes, as reflected in predicted upstream functional pathway analysis. CONCLUSION: The miR expression pattern in BM HSPCs after PT (+/-PNA) is dissimilar in old versus young adult mice. In the acute postinjury phase, old adult mice are unable to mount a robust miR HSPC response. Hematopoietic stem and progenitor cell miR expression in old PT mice reflects a diminished functional status and a blunted capacity for terminal differentiation of myeloid cells.

Performance Improvement With Implementation of a Surgical Skills Curriculum.

OBJECTIVE: To assess the efficacy of an intern surgical skills curriculum involving a boot camp for core open and laparoscopic skills, self-guided practice with positive and negative incentives, and semiannual performance evaluations. DESIGN: Longitudinal cohort study. SETTING: Academic tertiary care center. PARTICIPANTS: Intervention group (n = 15): residents who completed the intern surgical skills curriculum and had performance evaluations in fall of intern year, spring of intern year, and fall of second year. Control group (n = 8): second-year residents who were 1 year ahead of the intervention group in the same residency program, did not participate in the curriculum, and had performance evaluations in fall of second year. RESULTS: In fall of second year of residency, the intervention group had better performance (presented as median values with interquartile ranges) than the control group on one-hand ties (left hand: 9.1 [6.3-10.1] vs 14.6 [13.5-15.4] seconds, p = 0.007; right hand: 8.7 [8.5-9.6] vs 11.5 [9.9-16.8] seconds, p = 0.039). The intervention group also had better performance on all open suturing skills, including mattress suturing (vertical: 33.4 [30.0-40.0] vs 55.8 [50.0-67.6] seconds, p = 0.001; horizontal: 28.7 [27.3-39.9] vs 52.7 [40.7-57.8] seconds, p = 0.003), and a water-filled glove clamp, divide, and ligate task (28.0 [25.0-31.0] vs 59.1 [53.0-93.0] seconds, p < 0.001). Finally, the intervention group had better performance on all laparoscopic skills, including peg transfer (66.0 [59.0-82.0] vs 95.2 [87.5-101.5] seconds, p = 0.018), circle cut (82.0 [69.0-124.0] seconds vs 191.8 [155.5-231.5] seconds, p = 0.002), and intracorporeal suturing (195.0 [117.0-200.0] seconds vs 359.5 [269.0-450.0] seconds, p = 0.002). CONCLUSIONS: Implementation of a comprehensive surgical skills curriculum was associated with improved performance on core open and laparoscopic skills. Further research is needed to understand and optimize motivational factors for deliberate practice and surgical skill acquisition.

Modulation of the HGF/c-Met Axis Impacts Prolonged Hematopoietic Progenitor Mobilization Following Trauma and Chronic Stress.

BACKGROUND: Trauma and hemorrhagic shock trigger mobilization of hematopoietic progenitor cells (HPC) from bone marrow to peripheral blood. Hepatocyte growth factor (HGF), tyrosine-protein kinase Met (c-Met), matrix metallopeptidase 9 (MMP-9), and corticosterone regulate this mobilization process. We hypothesized that beta-blockade with propranolol and sympathetic outflow inhibition with clonidine following trauma and chronic stress would decrease hematopoietic progenitor cell mobilization. METHODS: Sprague-Dawley rats were randomized to undergo three models of injury and stress: lung contusion, LC plus hemorrhagic shock (LCHS), or LCHS plus chronic restraint stress for 2 h daily (LCHS/CS). Propranolol and clonidine were administered by daily intraperitoneal injection until sacrifice on day seven. Bone marrow HGF, c-Met, and MMP-9 were measured by real-time PCR. Plasma corticosterone was measured by ELISA. Percentage HPC in peripheral blood was measured by flow cytometry. RESULTS: Propranolol and clonidine significantly decreased bone marrow MMP-9 expression, plasma corticosterone levels, and HPC mobilization, and significantly increased hemoglobin levels. HPC mobilization was greatest following LCHS/CS (5.4 ±â€Š1.8) and was significantly decreased by propranolol (2.2 ±â€Š0.9, P < 0.001) and clonidine (1.7 ±â€Š0.5, P < 0.001). Hemoglobin (g/dL) was lowest following LCHS/CS (12.3 ±â€Š1.2) and was significantly increased by propranolol (13.7 ±â€Š0.4, P = 0.022) and clonidine (14.1 ±â€Š1.1, P < 0.001). CONCLUSIONS: Severe injury was associated with increased bone marrow HGF, c-Met, and MMP-9, circulating corticosterone, HPC mobilization, and persistent anemia. Attenuating the neuroendocrine response to injury and stress with propranolol and clonidine reduced MMP-9 expression, corticosterone levels, HPC mobilization, and the degree of anemia.

Postoperative surgical trainee opioid prescribing practices (POST-OPP): A national survey.

BACKGROUND: Increasing opioid-related deaths have heightened focus on combating the opioid epidemic. The impact of surgical trainees on opioid-related deaths is unclear, and there is little data examining the association between trainee pain management education and opioid prescribing practices. METHODS: An anonymous, online survey was distributed to members of the Resident and Associate Society of the American College of Surgeons. The survey covered five themes: education and knowledge, prescribing practices, clinical case scenarios, policy, and beliefs and attitudes. Linear mixed models were used to evaluate the influence of respondent characteristics on reported morphine milligram equivalents (MME) prescribed for common general surgery clinical scenarios. RESULTS: Of 427 respondents, 54 percent indicated receiving training in postoperative pain management during medical school and 66 percent during residency. Only 35 percent agreed that they had received adequate training in prescribing opioids. There was a significant association between undergoing formal pain management training in medical school and prescribing fewer MME for common outpatient general surgery scenarios (94 ± 15.2 vs 108 ± 15.0; p = 0.003). Similarly, formal pain management training in residency was associated with prescribing fewer MME in the survey scenarios (92.6 ± 15.2 vs 109 ± 15.2; p = 0.002). CONCLUSION: In this survey, nearly two-thirds of surgical residents felt that they were inadequately trained in opioid pre-scribing. Our findings additionally suggest that improving education may result in increased resident comfort with man-aging surgical pain, potentially leading to more responsible opioid prescribing. Further work will facilitate residency pro-grams' development of educational curricula for opioid prescribing best practices.

Persistent inflammation and anemia among critically ill septic patients.

BACKGROUND: Associations among inflammatory cytokines, erythropoietin (EPO), and anemia in critically ill septic patients remain unclear. This study tested the hypothesis that elevated inflammatory cytokines and decreased EPO would be associated with iron-restricted anemia while accounting for operative blood loss, phlebotomy blood loss, and red blood cell (RBC) transfusion volume. METHODS: Prospective observational cohort study of 42 critically ill septic patients was conducted. Hemoglobin (Hb) at sepsis onset and hospital discharge were used to calculate ΔHb. Operative blood loss, phlebotomy blood loss, and RBC transfusion volume were used to calculate adjusted ΔHb (AdjΔHb) assuming that 300 mL RBC is equal to 1 g/dL Hb. Patients with AdjΔHb of greater than 0 (positive AdjΔHb, n = 18) were compared with patients with AdjΔHb of less than or equal to 0 (negative AdjΔHb, n = 24). RESULTS: Plasma tumor necrosis factor α, granulocyte colony-stimulating factor, interleukin (IL)-6, IL-8, EPO, erythrocyte mean corpuscular volume, and serum transferrin receptor were measured on days 0, 1, 4, 7, and 14. Patients with negative AdjΔHb had significantly higher day 14 levels of IL-6 (37.4 vs. 15.2 pg/mL, p < 0.05), IL-8 (39.1 vs. 18.2 pg/mL, p = 0.01), and granulocyte colony-stimulating factor (101.3 vs. 60.5 pg/mL, p = 0.01), but not EPO. On linear regression analysis, lower AdjΔHb was associated with higher day 14 levels of IL-6 (r = 0.22, p < 0.01), IL-8 (r = 0.10, p = 0.04), stromal cell-derived factor 1 (r = 0.14, p = 0.02), and tumor necrosis factor α (r = 0.13, p = 0.02), but not EPO. Patients with negative AdjΔHb had significantly lower mean corpuscular volume on days 4 (89.6 vs. 93.2 fL/cell, p = 0.04), 7 (92.3 vs. 94.9 fL/cell, p = 0.04), and 14 (92.1 vs. 96.0 fL/cell, p = 0.03) but similar serum transferrin receptor levels. CONCLUSION: Persistent elevation of inflammatory cytokines was associated with iron-restricted anemia among critically ill septic patients, occurring in the absence of systemic iron deficiency, independent of endogenous EPO. LEVEL OF EVIDENCE: Prognostic study, level II.

Postoperative surgical trainee opioid prescribing practices (POST OPP): an institutional study.

BACKGROUND: Increasing mortality from opioid overdoses has prompted increased focus on prescribing practices of physicians. Unfortunately, resident physicians rarely receive formal education in effective opioid prescribing practices or postoperative pain management. Data to inform surgical training programs regarding the utility and feasibility of formal training are lacking. METHODS: Following Institutional Review Board approval, a single institution's resident physicians who had completed at least one surgical rotation were surveyed to assess knowledge of pain management and evaluate opioid prescribing practices. RESULTS: Fifty-three respondents (68% males and 32% females) completed the survey. Most respondents denied receiving formal instruction in opioid pain medication prescribing practices during either medical school (62.3%) or residency (56.6%); however, nearly all respondents stated they were aware of the side effects of opioid pain medications, and a majority felt confident in their knowledge of opioid pharmacokinetics and pharmacodynamics. Of the respondents, 47% either "agreed" or "strongly agreed" that they prescribed more opioid medications than necessary to patients being discharged following a surgical procedure. Individual case scenario responses demonstrated variability in the number of morphine milligram equivalents prescribed across scenarios (P < 0.001). Male and nonsurgical specialty respondents reported prescribing significantly fewer overall morphine milligram equivalents in these scenarios. CONCLUSIONS: This pilot study shows wide variability in opioid prescribing practices and attitudes toward pain management among surgical trainees, illustrating the potential utility of formal education in pain management and effective prescribing of these medications. A broader assessment of surgical trainees' knowledge and perception of opioid prescribing practices is warranted to facilitate the development of such a program.

The Postinjury Inflammatory State and the Bone Marrow Response to Anemia.

RATIONALE: The pathophysiology of persistent injury-associated anemia is incompletely understood, and human data are sparse. OBJECTIVES: To characterize persistent injury-associated anemia among critically ill trauma patients with the hypothesis that severe trauma would be associated with neuroendocrine activation, erythropoietin dysfunction, iron dysregulation, and decreased erythropoiesis. METHODS: A translational prospective observational cohort study comparing severely injured, blunt trauma patients who had operative fixation of a hip or femur fracture (n = 17) with elective hip repair patients (n = 22). Bone marrow and plasma obtained at the index operation were assessed for circulating catecholamines, systemic inflammation, erythropoietin, iron trafficking pathways, and erythroid progenitor growth. Bone marrow was also obtained from healthy donors from a commercial source (n = 8). MEASUREMENTS AND MAIN RESULTS: During admission, trauma patients had a median of 625 ml operative blood loss and 5 units of red blood cell transfusions, and Hb decreased from 10.5 to 9.3 g/dl. Compared with hip repair, trauma patients had higher median plasma norepinephrine (21.9 vs. 8.9 ng/ml) and hepcidin (56.3 vs. 12.2 ng/ml) concentrations (both P < 0.05). Bone marrow erythropoietin and erythropoietin receptor expression were significantly increased among patients undergoing hip repair (23% and 14% increases, respectively; both P < 0.05), but not in trauma patients (3% and 5% increases, respectively), compared with healthy control subjects. Trauma patients had lower bone marrow transferrin receptor expression than did hip repair patients (57% decrease; P < 0.05). Erythroid progenitor growth was decreased in trauma patients (39.0 colonies per plate; P < 0.05) compared with those with hip repair (57.0 colonies per plate; P < 0.05 compared with healthy control subjects) and healthy control subjects (66.5 colonies per plate). CONCLUSIONS: Severe blunt trauma was associated with neuroendocrine activation, erythropoietin dysfunction, iron dysregulation, erythroid progenitor growth suppression, and persistent injury-associated anemia. Clinical trial registered with www.clinicaltrials.gov (NCT 02577731).

Persistent injury-associated anemia and aging: Novel insights.

BACKGROUND: Hypercatecholaminemia and bone marrow dysfunction have been implicated in the pathophysiology of persistent injury-associated anemia. The elderly may be more vulnerable to bone marrow dysfunction due to high basal and peak catecholamine levels and impaired hematopoietic progenitor growth. We hypothesized that aging would adversely affect persistent injury-associated anemia. METHODS: Male Sprague-Dawley rats aged 8 to 9 weeks and F344-BN rats aged 25 months were randomized to naive controls, lung contusion plus hemorrhagic shock (LCHS), and LCHS plus daily chronic restraint stress (LCHS/CS). Urine norepinephrine was measured on Days 1 and 7. Mobilization of hematopoietic progenitor cells (HPCs), bone marrow colony-forming units-erythroid growth, and peripheral blood hemoglobin, mean corpuscular volume (MCV), and red cell distribution width (RDW) were assessed on Day 7 (*p < 0.05 young vs. aged counterpart by one-way analysis of variance). RESULTS: Aged rats had higher norepinephrine levels at naive baseline (97* vs. 27 ng/mL) and 7 days following LCHS/CS when compared with young (359* vs. 127 ng/mL). Following LCHS/CS, HPC mobilization was greater among young rats when compared with aged (5.4 vs. 2.5%). Colony-forming units-erythroid growth was lower among aged animals for each group (naive: 47* vs. 65; LCHS: 40* vs. 50; LCHS/CS: 38* vs. 44 cells/plate). Aged naive rats had higher initial hemoglobin (15.2* vs. 14.3 g/dL) but lower MCV (48* vs. 59 fL/cell) and larger RDW at baseline and greater differences 7 days after LCHS/CS (MCV: 46* vs. 60 fL/cell; RDW: 17.4* vs. 16.3%). CONCLUSIONS: Compared with young rats, aged rats had less HPC mobilization despite elevated basal and peak norepinephrine. Aged rats were disproportionately affected by impaired hematopoietic progenitor growth and an iron-restricted red blood cell phenotype at baseline, which persisted 7 days after injury. Further research is needed to assess how the clinical approach to persistent injury-associated anemia should differ for elderly trauma patients.

Persistent injury-associated anemia in aged rats.

BACKGROUND: Hypercatecholaminemia and bone marrow dysfunction have been implicated in the pathophysiology of persistent-injury associated anemia. The elderly may be vulnerable to this phenomenon due to high basal and peak catecholamine levels, impaired erythroid progenitor growth, and baseline anemia. We hypothesized that aged F344-BN rats subjected to severe trauma and chronic stress would have persistent injury-associated anemia. METHODS: Male F344-BN rats age 25months were randomly allocated to: naïve (n=8), lung contusion (LC, n=9), LC followed by daily chronic restraint stress (LC/CS, n=9), LC followed immediately by hemorrhagic shock (LCHS, n=8), and LCHS followed by daily CS (LCHS/CS, n=8). Urine norepinephrine was measured on days one and seven. Locomotor testing was performed on day five. Bone marrow cellularity, hematopoietic progenitor growth, and peripheral blood hemoglobin levels were assessed at sacrifice on day seven. Data are presented as mean±standard deviation, *p<0.05 vs. naïve. RESULTS: Norepinephrine levels (ng/mL) were significantly elevated one day after LCHS (420±239* vs. naïve: 97±71) and LCHS/CS (375±185*), and remained significantly elevated on day seven for LCHS/CS (359±99*), but not LCHS (212±130). On locomotor testing, groups subjected to CS traveled shorter distances at lower velocities and spent less time in the center of the cage. Colony forming units-erythroid (colonies/plate), representing late erythroid progenitors, were significantly decreased after LC/CS (40±1* vs. naïve: 47±4), LCHS (40±1*), and LCHS/CS (38±3*). LCHS/CS animals had significantly lower hemoglobin (g/dL) than naïve animals (13.3±1.3* vs. naïve: 15.2±0.9). CONCLUSIONS: Persistent injury-associated anemia occurs in aged rats. Further research is needed to determine whether the pathophysiology of this phenomenon differs from that of younger rats, and to translate these findings to elderly trauma patients.

Benchmarking clinical outcomes and the immunocatabolic phenotype of chronic critical illness after sepsis in surgical intensive care unit patients.

BACKGROUND: A growing number of patients survive sepsis but remain chronically critically ill. We sought to define clinical outcomes and incidence of chronic critical illness (CCI) after sepsis and to determine whether selected biomarkers of inflammation, immunosuppression, and catabolism differ between these patients and those that rapidly recover (RAP). METHODS: This 3-year prospective observational cohort study (NCT02276417) evaluated 145 surgical intensive care unit patients with sepsis for the development of CCI (≥14 days of intensive care unit resource utilization with persistent organ dysfunction). Patient clinical demographics, outcomes, and serial serum/urine samples were collected for plasma protein and urinary metabolite analyses. RESULTS: Of 145 sepsis patients enrolled, 19 (13%) died during their hospitalization and 71 (49%) developed CCI. The CCI patients were significantly older (mean, 63 ± 15 vs. 58 ± 13 years, p = 0.006) and more likely to be discharged to long-term acute care facilities (32% vs. 3%, p < 0.0001), whereas those with RAP were more often discharged to home or a rehabilitation facility. Six-month mortality was significantly higher in CCI as compared with RAP cohort (37% vs. 2%; p < 0.01). Multivariate logistic regression modeling revealed delayed onset sepsis (>48 hours after admission; odds ratio [OR], 10.93; 95% confidence interval [CI], 4.15-28.82]), interfacility transfer (OR, 3.58; 95% CI, 1.43-8.96), vasopressor-dependent septic shock (OR, 3.75; 95% CI, 1.47-9.54), and Sequential Organ Failure Assessment score of 5 or greater at 72 hours (OR, 5.03; 95% CI, 2.00-12.62) as independent risk factors for the development of CCI. The CCI patients also demonstrated greater elevations in inflammatory cytokines (IL-6, IL-8, IL-10), and biomarker profiles are consistent with persistent immunosuppression (absolute lymphocyte count and soluble programmed death ligand 1) and catabolism (plasma insulin-like growth factor binding protein 3 and urinary 3-methylhistidine excretion). CONCLUSION: The development of CCI has become the predominant clinical trajectory in critically ill surgical patients with sepsis. These patients exhibit biomarker profiles consistent with an immunocatabolic phenotype of persistent inflammation, immunosuppression, and catabolism. LEVEL OF EVIDENCE: Prognostic, level II.

Sepsis and Critical Illness Research Center investigators: protocols and standard operating procedures for a prospective cohort study of sepsis in critically ill surgical patients.

INTRODUCTION: Sepsis is a common, costly and morbid cause of critical illness in trauma and surgical patients. Ongoing advances in sepsis resuscitation and critical care support strategies have led to improved in-hospital mortality. However, these patients now survive to enter state of chronic critical illness (CCI), persistent low-grade organ dysfunction and poor long-term outcomes driven by the persistent inflammation, immunosuppression and catabolism syndrome (PICS). The Sepsis and Critical Illness Research Center (SCIRC) was created to provide a platform by which the prevalence and pathogenesis of CCI and PICS may be understood at a mechanistic level across multiple medical disciplines, leading to the development of novel management strategies and targeted therapies. METHODS: Here, we describe the design, study cohort and standard operating procedures used in the prospective study of human sepsis at a level 1 trauma centre and tertiary care hospital providing care for over 2600 critically ill patients annually. These procedures include implementation of an automated sepsis surveillance initiative, augmentation of clinical decisions with a computerised sepsis protocol, strategies for direct exportation of quality-filtered data from the electronic medical record to a research database and robust long-term follow-up. ETHICS AND DISSEMINATION: This study has been registered at ClinicalTrials.gov, approved by the University of Florida Institutional Review Board and is actively enrolling subjects. Dissemination of results is forthcoming.

Persistent Inflammation, Immunosuppression and Catabolism Syndrome.

Following advances in critical care, in-hospital multiple organ failure-related mortality is declining. Consequently, incidence of chronic critical illness is increasing. These patients linger in the intensive care unit, have high resource utilization, and poor long-term outcomes. Within this population, the authors propose that a substantial subset of patients have a new phenotype: persistent inflammation, immunosuppression, and catabolism syndrome. There is evidence that myelodysplasia with expansion of myeloid-derived suppressor cells, innate and adaptive immune suppression, and protein catabolism with malnutrition are major contributors. Optimal care of these patients will require novel multimodality interventions.

Antibody-specific detection of CAIX in breast and prostate cancers.

Carbonic anhydrase IX (CAIX) is frequently expressed in human tumors and serves as a marker for hypoxia. Further, CAIX expression is considered a predictor of poor survival in many, but not all, cancer types. Herein, we compare the specificity of two CAIX antibodies: the M75, monoclonal antibody which recognizes an epitope in the N-terminus and a commercially available polyclonal antibody generated against a C-terminal peptide (NB100-417). Western blot analysis of multiple breast cell lines revealed that the polyclonal antibody detected both membrane-bound and soluble proteins. The M75 antibody recognized only the membrane-bound species, which is presumed to be CAIX. These data were confirmed in an aggressive prostate cell line. We further compared these antibodies in prostate tumors by immunohistochemistry. Staining with NB100 was comparable to that of the M75 antibody, but only at high dilution. Otherwise, cytoplasmic staining was also noted. Two-dimensional gel electrophoresis followed by mass spectrometric analysis revealed that the cytoplasmic protein detected by NB100 is beta-tubulin. This cross-reactivity could lead to false-positives for CAIX expression in samples where cytosolic proteins are present.