Hew D. T. Torrance

Perioperative blood transfusion is associated with a gene transcription profile characteristic of immunosuppression: a prospective cohort study

IntroductionBlood transfusion in the perioperative period has frequently been associated with an excess of nosocomial infections. Whilst transfused whole blood induces specific host immune alteration that may predispose to nosocomial infections, the immunomodulating properties associated with leukodepleted blood remain incompletely understood. In this study, we explore the hypothesis that the transfusion of leukodepleted allogeneic blood during or following major gastrointestinal surgery is associated with an immunosuppressed phenotype, which may in turn predispose to postoperative infectious complications.MethodsPatients aged over 45 years undergoing scheduled inpatient major gastrointestinal surgery were recruited. Gene expression profiles of specific inflammatory genes were assayed from blood collected preoperatively, at 24 and at 48 hours after surgery. Genes were selected based on their ability to represent specific immune pathways. Gene expression was quantified using quantitative real-time polymerase chain reaction (qRT-PCR) to measure messenger RNA (mRNA) levels. Postoperative infections were documented using predefined criteria.ResultsOne hundred and nineteen patients were recruited. Fifteen (13%) patients required blood transfusion within 24 hours of surgery, 44 (37%) patients developed infections and 3 (2%) patients died prior to discharge. Patients receiving a blood transfusion were more likely to develop postoperative infections (P =0.02) and to have lower tumour necrosis factor alpha (TNFα), interleukin (IL)-12, IL-23 and RAR-related orphan receptor gamma T (RORγt) gene expression in the postoperative period (P <0.05). The TNFα/IL-10 mRNA ratio at 24 hours (P =0.0006) and at 48 hours (P =0.01) was lower in patients receiving a blood transfusion over this period. Multivariable analysis confirmed that these observations were independent of the severity of the surgical insult.ConclusionsAn association between an immunosuppressive pattern of gene expression and blood transfusion following major elective gastrointestinal surgery is described. This gene expression profile includes a reduction in the activity of innate immunity and T helper cell type 1 (Th1) and T helper cell type 17 (Th17) pathways in those patients receiving a blood transfusion. Blood transfusion was also associated with an excess of infectious complications in this cohort. A mechanistic link is suggested but not proven.

A single-centre observational cohort study of admission National Early Warning Score (NEWS).

INTRODUCTION Early warning scores are commonly used in hospitals to identify patients at risk of deterioration. The National Early Warning Score (NEWS) has recently been introduced to UK practice. However, it is not yet widely implemented. We aimed to compare NEWS to the early warning score currently used in our hospital--the Patient at Risk Score (PARS). METHODS We conducted a prospective observational cohort study of all adult general medical patients admitted to a single hospital over a 20-day period. Physiological data and early warning scores recorded in bedside charts were collected on admission and a NEWS score was retrospectively calculated. The patient notes were reviewed at 48 h after admission. The primary outcome was a composite of critical care admission or death within 2 days of admission. The secondary outcome was hospital length of stay. RESULTS NEWS was more strongly associated with the primary outcome than PARS (odds ratio 1.54, p < 0.001 compared to 1.42, p = 0.056). A NEWS of 3 or more was associated with the primary outcome (odds ratio 7.03, p = 0.003). Neither score was correlated with hospital length of stay. CONCLUSION NEWS on admission is superior to PARS for identifying patients at risk of death or critical care admission within the first 2 days of hospital stay. Current guidelines advocate a threshold of 5 for triggering a clinical review. However, since a score of 3 or more was associated with a poor outcome, this recommendation should be reviewed. Both scores were poor predictors of hospital length of stay.

The perioperative immune response.

Purpose of reviewA host of immune modulators are now available in clinical practice. The perioperative period is characterized by profound alterations in host immunity, which can result in poor outcomes, which include infection, cancer recurrence and organ failure. Manipulation of the perioperative immune response has the potential to improve outcomes. A complete understanding of the mechanisms and clinical consequences of altered immune function in this setting is therefore imperative. Recent findingsRecent in-vivo data have emerged which further our understanding of the interaction between tissue damage, immune modulation and clinical outcomes by utilizing novel laboratory techniques capable of monitoring single-cell immune signatures. Traditional gene expression assays have continued to demonstrate their utility and have been instrumental in defining the host response to perioperative allogeneic blood transfusion. These mechanistic studies are complemented by large clinical studies describing associations between anaesthetic modalities and immune-related outcomes. SummaryLaboratory techniques are now available that can monitor the perioperative immune response and could be further developed to introduce personalized care pathways. Consideration must also be given to anaesthesia techniques and perioperative treatments that, although not immediately harmful, may be associated with poor outcomes temporally distant from the treatment, secondary to induced immunosuppression.

Systemic inflammatory response syndrome after major abdominal surgery predicted by early upregulation of TLR4 and TLR5

Objectives:To study innate immune pathways in patients undergoing hepatopancreaticobiliary surgery to understand mechanisms leading to enhanced inflammatory responses and identifying biomarkers of adverse clinical consequences. Background:Patients undergoing major abdominal surgery are at risk of life-threatening systemic inflammatory response syndrome (SIRS) and sepsis. Early identification of at-risk patients would allow tailored postoperative care and improve survival. Methods:Two separate cohorts of patients undergoing major hepatopancreaticobiliary surgery were studied (combined n = 69). Bloods were taken preoperatively, on day 1 and day 2 postoperatively. Peripheral blood mononuclear cells and serum were separated and immune phenotype and function assessed ex vivo. Results:Early innate immune dysfunction was evident in 12 patients who subsequently developed SIRS (postoperative day 6) compared with 27 who did not, when no clinical evidence of SIRS was apparent (preoperatively or days 1 and 2). Serum interleukin (IL)-6 concentration and monocyte Toll-like receptor (TLR)/NF-&kgr;B/IL-6 functional pathways were significantly upregulated and overactive in patients who developed SIRS (P < 0.0001). Interferon &agr;-mediated STAT1 phosphorylation was higher preoperatively in patients who developed SIRS. Increased TLR4 and TLR5 gene expression in whole blood was demonstrated in a separate validation cohort of 30 patients undergoing similar surgery. Expression of TLR4/5 on monocytes, particularly intermediate CD14++CD16+ monocytes, on day 1 or 2 predicted SIRS with accuracy 0.89 to 1.0 (areas under receiver operator curves). Conclusions:These data demonstrate the mechanism for IL-6 overproduction in patients who develop postoperative SIRS and identify markers that predict patients at risk of SIRS 5 days before the onset of clinical signs.

Changes in gene expression following trauma are related to the age of transfused packed red blood cells.

BACKGROUND Transfusion of packed red blood cells (PRBCs) is associated with an increased incidence of nosocomial infections and an increased risk of death. The duration of storage before transfusion may influence these outcomes. Here, we explore the association between the age of transfused PRBCs and specific patterns of inflammatory gene expression in severely injured trauma patients. METHODS Severely injured trauma patients requiring intensive care unit treatment and receiving transfusion of PRBCs within 24 hours of the injury were recruited. Blood samples were obtained within 2 hours of the trauma, at 24 hours, and at 72 hours. Messenger RNA was extracted from whole blood, and gene expression was quantified using quantitative polymerase chain reaction. The median age of the units of PRBCs transfused to each patient was recorded. The primary outcome measure was the change in candidate gene expression over the initial 72 hours. RESULTS Sixty-four patients were studied. Fifty-three patients (83%) were male, and the median age was 40.5 years (interquartile range [IQR], 31–59). Median Injury Severity Score (ISS) was 31.5 (IQR, 23–43), and 55 patients (86%) experienced a blunt injury. Forty-one patients (64%) developed a nosocomial infection, and 15 patients (23%) died before hospital discharge. Each patient received a median of 5 U of PRBCs (IQR, 4–9.8 U) during the first 24 hours of hospital admission. The median age of the units of PRBCs transfused in each patient was 20 days (IQR, 17–22 days). Older blood was associated with greater decreases in interleukin 12 (IL-12), IL-23, and ROR&ggr;t (all p’s < 0.05) gene expression over the initial 24 hours, greater decreases in IL-12 gene expression over 72 hours, and a rise in transforming growth factor &bgr; gene expression over the first 72 hours. A multivariate analysis confirmed the independence of these associations. CONCLUSION Increasing the duration of storage of PRBCs before transfusion is associated with a pattern of gene expression consistent with more severe immunosuppression. LEVEL OF EVIDENCE Epidemiologic study, level III.

Artesunate Protects Against the Organ Injury and Dysfunction Induced by Severe Hemorrhage and Resuscitation.

OBJECTIVE To evaluate the effects of artesunate on organ injury and dysfunction associated with hemorrhagic shock (HS) in the rat. BACKGROUND HS is still a common cause of death in severely injured patients and is characterized by impairment of organ perfusion, systemic inflammatory response, and multiple organ failure. There is no specific therapy that reduces organ injury/dysfunction. Artesunate exhibits pharmacological actions beyond its antimalarial activity, such as anticancer, antiviral, and anti-inflammatory effects. METHODS Rats were submitted to HS. Mean arterial pressure was reduced to 30 mm Hg for 90 minutes, followed by resuscitation. Rats were randomly treated with artesunate (2.4 or 4.8 mg/kg i.v.) or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed. RESULTS Artesunate attenuated the multiple organ injury and dysfunction caused by HS. Pathway analysis of RNA sequencing provided good evidence to support an effect of artesunate on the Akt-survival pathway, leading to downregulation of interleukin-1 receptor-associated kinase 1. Using Western blot analysis, we confirmed that treatment of HS rats with artesunate enhanced the phosphorylation (activation) of Protein kinase B (Akt) and endothelial nitric oxide synthase and the phosphorylation (inhibition) of glycogen synthase kinase-3β (GSK-3β). Moreover, artesunate attenuated the HS-induced activation of nuclear factor kappa B and reduced the expression of proinflammatory proteins (inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin 6). CONCLUSIONS Artesunate attenuated the organ injury/dysfunction associated with HS by a mechanism that involves the activation of the Akt-endothelial nitric oxide synthase survival pathway, and the inhibition of glycogen synthase kinase-3β and nuclear factor kappa B. A phase II clinical trial evaluating the effects of good manufacturing practice-artesunate in patients with trauma and severe hemorrhage is planned.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives 4.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. http://creativecommons.org/licenses/by-nc-nd/4.0.Objective: To evaluate the effects of artesunate on organ injury and dysfunction associated with hemorrhagic shock (HS) in the rat. Background: HS is still a common cause of death in severely injured patients and is characterized by impairment of organ perfusion, systemic inflammatory response, and multiple organ failure. There is no specific therapy that reduces organ injury/dysfunction. Artesunate exhibits pharmacological actions beyond its antimalarial activity, such as anticancer, antiviral, and anti-inflammatory effects. Methods: Rats were submitted to HS. Mean arterial pressure was reduced to 30 mm Hg for 90 minutes, followed by resuscitation. Rats were randomly treated with artesunate (2.4 or 4.8 mg/kg i.v.) or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed. Results: Artesunate attenuated the multiple organ injury and dysfunction caused by HS. Pathway analysis of RNA sequencing provided good evidence to support an effect of artesunate on the Akt-survival pathway, leading to downregulation of interleukin-1 receptor-associated kinase 1. Using Western blot analysis, we confirmed that treatment of HS rats with artesunate enhanced the phosphorylation (activation) of Protein kinase B (Akt) and endothelial nitric oxide synthase and the phosphorylation (inhibition) of glycogen synthase kinase-3&bgr; (GSK-3&bgr;). Moreover, artesunate attenuated the HS-induced activation of nuclear factor kappa B and reduced the expression of proinflammatory proteins (inducible nitric oxide synthase, tumor necrosis factor-&agr;, and interleukin 6). Conclusions: Artesunate attenuated the organ injury/dysfunction associated with HS by a mechanism that involves the activation of the Akt-endothelial nitric oxide synthase survival pathway, and the inhibition of glycogen synthase kinase-3&bgr; and nuclear factor kappa B. A phase II clinical trial evaluating the effects of good manufacturing practice-artesunate in patients with trauma and severe hemorrhage is planned.

A single-centre cohort study of National Early Warning Score (NEWS) and near patient testing in acute medical admissions.

INTRODUCTION The utility of an early warning score may be improved when used with near patient testing. However, this has not yet been investigated for National Early Warning Score (NEWS). We hypothesised that the combination of NEWS and blood gas variables (lactate, glucose or base-excess) was more strongly associated with clinical outcome compared to NEWS alone. METHODS This was a prospective cohort study of adult medical admissions to a single-centre over 20days. Blood gas results and physiological observations were recorded at admission. NEWS was calculated retrospectively and combined with the biomarkers in multivariable logistic regression models. The primary outcome was a composite of mortality or critical care escalation within 2days of hospital admission. The secondary outcome was hospital length of stay. RESULTS After accounting for missing data, 15 patients out of 322 (4.7%) died or were escalated to the critical care unit. The median length of stay was 4 (IQR 7) days. When combined with lactate or base excess, NEWS was associated with the primary outcome (OR 1.18, p=0.01 and OR 1.13, p=0.03). However, NEWS alone was more strongly associated with the primary outcome measure (OR 1.46, p<0.01). The combination of NEWS with glucose was not associated with the primary outcome. Neither NEWS nor any combination of NEWS and a biomarker were associated with hospital length of stay. CONCLUSION Admission NEWS is more strongly associated with death or critical care unit admission within 2days of hospital admission, compared to combinations of NEWS and blood-gas derived biomarkers.

Features of Postoperative Immune Suppression Are Reversible With Interferon Gamma and Independent of Interleukin-6 Pathways.

Objective: The aim of this study was to evaluate the role of interleukin (IL)-6 pathways in postoperative immune suppression and to assess the reversibility of this phenomenon. Background: The postoperative period is characterized by increased IL-6 production and features of immune suppression. In vitro, IL-6 mediates anti-inflammatory effects through inhibition of interferon gamma (IFN-&ggr;) pathways. The significance of the immunomodulatory effects of IL-6 in the clinical setting of postoperative immune suppression remains unclear. Methods: Patients over 45 years old undergoing elective surgery, involving the gastrointestinal tract, were recruited. IL-6 levels were assayed using an enzyme linked immunosorbent assay preoperatively, and at 24 and 48 hours. Peripheral blood mononuclear cells from healthy volunteers were cultured in perioperative serum and CD14+Human Leukocyte Antigen-DR (HLA-DR) [monocyte HLA-DR (mHLA-DR)] geometric mean florescent intensity was measured in the presence and absence of IL-6 neutralizing antibody and recombinant IFN-&ggr;. Results: Of the 108 patients, 41 developed a postoperative infection. The IL-6 levels increased 19-fold from the preoperative sample to 24 hours postoperatively (P < 0.0001). Higher IL-6 levels at 24 (P = 0.0002) and 48 hours (P = 0.003) were associated with subsequent postoperative infectious complications. mHLA-DR mean florescent intensity fell when healthy peripheral blood mononuclear cells were cultured with postoperative serum compared with preoperative serum (P = 0.008). This decrease was prevented by the presence of IFN-&ggr; in the culture media, but not by the presence of IL-6-neutralizing antibody. Conclusions: IL-6 levels increase after a major surgery and are associated with an increased susceptibility to postoperative infections. Serum obtained from postoperative patients induces an immunosuppressive response, reflected in reduced mHLA-DR levels, mediated through IL-6 independent pathways and is reversible with IFN-&ggr;. These data may have therapeutic implications for the prevention of infection in patients undergoing major surgery.

Association between gene expression biomarkers of immunosuppression and blood transfusion in severely injured polytrauma patients

Objective: To explore the hypothesis that blood transfusion contributes to an immunosuppressed phenotype in severely injured patients. Background: Despite trauma patients using disproportionately large quantities of blood and blood products, the immunomodulatory effects of blood transfusion in this group are inadequately described. Methods: A total of 112 ventilated polytrauma patients were recruited. Messenger RNA (mRNA) was extracted from PAXGene tubes collected within 2 hours of the trauma, at 24 hours, and at 72 hours. T-helper cell subtype specific cytokines and transcription factors were quantified using real-time polymerase chain reaction. Results: Median injury severity score was 29. Blood transfusion was administered to 27 (24%) patients before the 2-hour sampling point. Transfusion was associated with a greater immediate rise in IL-10 (P = 0.003) and IL-27 (P = 0.04) mRNA levels. Blood products were transfused in 72 (64%) patients within the first 24 hours. There was an association between transfusion at 24 hours and higher IL-10 (P < 0.0001), lower Foxp3 (P = 0.01), GATA3 (P = 0.006), and RORγ t( P = 0.05) mRNA levels at 24 hours. There were greater reductions in T-bet (P = 0.03) mRNA levels and lesser increases in TNFα (P = 0.015) and IFNγ (P = 0.035) at 24 hours in those transfused. Multiple regression models confirmed that the transfusion of blood products was independently associated with altered patterns of gene expression. Blood stream infections occur in 15 (20.8%) of those transfused in the first 24 hours, compared with 1 patient (2.5%) not transfused (OR = 10.3 [1.3–81], P = 0.008). Conclusions: The primarily immunosuppressive inflammatory response to polytrauma may be exacerbated by the transfusion of blood products. Furthermore, transfusion was associated with an increased susceptibility to nosocomial infections.

Epigenetic regulatory pathways involving microRNAs may modulate the host immune response following major trauma

BACKGROUND Posttraumatic nosocomial pneumonia is a common complication resulting in significant morbidity. Trauma-induced immunocompromise is associated with an enhanced susceptibility to pneumonia. In this study, we explore the hypothesis that posttranscriptional epigenetic regulation of gene expression may be an important factor in determining this immune phenotype. We describe the pattern of production of microRNAss (miRs) and their association with nosocomial pneumonia following severe trauma. METHODS A convenience sample of 30 ventilated polytrauma patients (UKCRN ID: 5637) and 16 healthy controls were recruited. Messenger RNA and protein levels of key cytokines were quantified within 2 hours of the injury and at 24 hours. Three miRs per cytokine were then selected based on miRBase target prediction scores and quantified using polymerase chain reaction. Nosocomial pneumonia was defined using the Centers for Disease Control and Prevention definitions. RESULTS Median Injury Severity Score (ISS) was 29, and 47% of the patients developed nosocomial pneumonia. miR-125a and miR-202 decreased by 34% and 77%, respectively, immediately following injury, whereas their target, IL-10, increased messenger RNA levels 3-fold and protein levels 180-fold. Tumor necrosis factor &agr; (TNF-&agr;) and IL-12 gene expression decreased by 68% and 43%, respectively, following injury, and this was mirrored by a 10-fold increase in miR-181, an miR predicted to target TNF-&agr; transcripts. Lower levels of miR-125a and miR-374b were associated with the later acquisition of hospital-acquired pneumonia. CONCLUSION Alteration in the expression of miRs with highly predicted complementarity to IL-10 and TNF-&agr; may be an important mechanism regulating the posttraumatic immunosuppressive phenotype in intensive care unit patients. LEVEL OF EVIDENCE Retrospective observational study, level III.