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 α (TNF-α) 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-α 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-α may be an important mechanism regulating the posttraumatic immunosuppressive phenotype in intensive care unit patients. LEVEL OF EVIDENCE Retrospective observational study, level III

T-helper cell polarisation following severe polytrauma

Introduction Severe polytrauma induces an immunosuppressive response and is associated with a very high incidence of nosocomial infections. Previous studies have inferred that this detrimental immune response results from polarisation of the T helper (Th) response towards an anti-inflammatory, TH2 dominated, response at the expense of a bactericidal, Th1 response [1]. Objectives 1) To define alterations in TH cell subsets following severe blunt polytrauma. Methods Patients presenting to the emergency department within 2 hours of severe polytrauma were eligible if intubated either at the scene or in ED. Isolated head injuries and those not expected to survive 24 hours were excluded. EDTA anti-coagulated blood was drawn at 0hr (within 2 hours of injury), at 24 and 72hrs. Samples were immediately lysed, washed, stained and analysed using a standardised human 8-colour TH 1, 2 & 17 panel [2] on an LSR II flow cytometer. A paired white cell count differential was obtained at each sampling point. Patients were followed until discharge or death. Data were analysed using non-parametric statistics, with results presented as median and IQR. Results 15 consecutive severe polytrauma patients requiring Intensive Care Unit (ICU) admission were recruited. Demographic and clinical data are outlined in Figure 1. Twelve (80%) lymphocytosis (3.3x109/L, 2.5 - 4.4x109/L) (Figyre 2A). At 72 hours leukocytes had fallen (P < 0.01, figure 2A) such that 6 (54%) of those surviving were lymphopenic (0.9x109/L, 0.6 - 1.2x109/L). Circulating CD4+ (P = 0.01; Figure 2B) and CD4+CD25+ (P < 0.05) lymphocytes increased over 72 hours. When expressed as a percentage of total circulating lymphocytes no significant change in the proportions of the TH 1, 2 & 17 subpopulations was detected (Figure 2C-E). Conclusions Severe polytrauma patients swiftly become lymphopenic. Although a failure to normalise this during the ICU stay correlates with higher mortality [3] our study of TH cell subtypes demonstrates no evidence of a switch to a detrimental anti-inflammatory TH2 subtype at the expense of the potentially protective bactericidal TH1 subtype

The role of micrornas in the development of hospital acquired infection in polytrauma patients

Introduction Traumatic injury is associated with immunosuppression and an increased risk of developing nosocomial infections. However, the immune regulatory mechanisms involved remain unclear. Objectives 1) To describe genome-wide alterations in micro RNA (miRNA) expression following severe trauma. 2) To explore the potential role of miRNAs in mediating the post-traumatic immunosuppressive phenotype and their potential role in enhancing the risk of nosocomial infections. Methods Patients requiring ICU care following traumatic injury were recruited. Whole blood was collected within 2 hours of injury and 24 hours later. Total RNA (containing miRNAs) was isolated utilising PAX Gene and RNA extraction kits (Qiagen). miRNA-sequencing was performed with the Illumina HiSeq2500, and sequences were aligned to the human GRCh37 reference genome. Data analysis was carried out using the DESEQ2 package in R, and miRNAs were considered significantly altered with an adjusted p value of < 0.05. Functional enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) on all miRNAs reaching an adjusted p value of < 0.1. mRNA targets of interest were identified using miRBase and TargetScan (http://www.mirbase.org, http://www.targetscan.org). Results 49 patients were recruited and 25 patients developed nosocomial infections. Expression of 139 miRNAs was significantly altered between 2 hours and 24 hours following injury, with miR-146b, a key inhibitor of pro-inflammatory pathways[1], upregulated to the greatest degree. Figure 1 presents miRNAs that differ between those patients who developed nosocomial infections and those who did not. miR-144-5p was significantly different between the two groups at both time points. a large percentage of mRNA targets for miR-144 are involved the Cell-mediated Immune Response (Figure 2), including the B-cell receptor complex, p38MAPK, GATA3, IgG, BCL6 and the T-cell receptor. in addition, we have previously shown that the miR-374 family of miRNAs is linked to increased IL-10 expression in trauma patients[2]. IPA highlights Cancer, Haematological Disease, Immunological and Inflammatory Disease and Organismal Injury and Abnormalities as important pathways altered between infected and non-infected patients. Conclusions These data provide a miRNA signature of severely injured trauma patients who develop hospital acquired infection compared to those who do not, and identify the miR-144 and miR-374b families as being of particular interest for future studies of trauma-induced immune dysfunction

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 RORγt (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 β 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

Immature platelet dynamics are associated with clinical outcomes after major trauma.

BACKGROUND: Major trauma results in dramatic changes in platelet behavior. Newly-formed platelets are more reactive than older platelets, but their contributions to hemostasis and thrombosis after severe injury have not been previously evaluated. OBJECTIVES: To determine the relationship between immature platelet metrics and plasma thrombopoietin with clinical outcomes after major injury. METHODS: Prospective observational cohort study of adult trauma patients. Platelet counts and the immature platelet fraction (IPF) were measured at admission, 24 hours, 72 hours and 7 days post-injury. Thromboelastometry was performed at admission. Plasma thrombopoietin, c-Mpl and GPIbα were quantified in a separate cohort. The primary outcome was in-hospital mortality; secondary outcomes were venous thromboembolic events (VTE) and multiple organ dysfunction (MODS). RESULTS: On admission, immature platelet counts (IPC) were significantly lower in non-survivors (n=40) compared to survivors (n=236; 7.3x109/L vs 10.6x109/L, p=0.009), but IPF did not differ. Similarly, impaired platelet function on thromboelastometry was associated with lower admission IPC (9.1x109/L vs 11.9x109/L, p<0.001). However, at later timepoints we observed significantly higher IPF and IPC in patients who developed VTE (21.0x109/L vs 11.1x109/L, p=0.02) and prolonged MODS (20.9 x109/L vs 11x109/L, p=0.003) compared to those who did not develop complications. Plasma thrombopoietin levels at admission were significantly lower in in non-survivors (p<0.001), in patients with MODS (p<0.001) and in those who developed VTE (p=0.04). CONCLUSIONS: Lower levels of immature platelets in the acute phase after major injury are associated with increased mortality, whereas higher immature platelet levels at later timepoints may predispose to thrombosis and multiple organ dysfunction

Comparative Analysis of V-Akt Murine Thymoma Viral Oncogene Homolog 3 (AKT3) Gene between Cow and Buffalo Reveals Substantial Differences for Mastitis

AKT3 gene is a constituent of the serine/threonine protein kinase family and plays a crucial role in synthesis of milk fats and cholesterol by regulating activity of the sterol regulatory element binding protein (SREBP). AKT3 is highly conserved in mammals and its expression levels during the lactation periods of cattle are markedly increased. AKT3 is highly expressed in the intestine followed by mammary gland and it is also expressed in immune cells. It is involved in the TLR pathways as effectively as proinflammatory cytokines. The aims of this study were to investigate the sequences differences between buffalo and cow. Our results showed that there were substantial differences between buffalo and cow in some exons and noteworthy differences of the gene size in different regions. We also identified the important consensus sequence motifs, variation in 2000 upstream of ATG, substantial difference in the “3′UTR” region, and miRNA association in the buffalo sequences compared with the cow. In addition, genetic analyses, such as gene structure, phylogenetic tree, position of different motifs, and functional domains, were performed to establish their correlation with other species. This may indicate that a buffalo breed has potential resistance to disease, environment changes, and airborne microorganisms and some good production and reproductive traits

Complication rates in managing hepatic trauma: a cross-sectional study stratifying their outcomes

Background: Liver trauma is the most commonly observed injured organ in abdominal trauma. The objectives of this study was to determine and evaluate the rates of complication in the management of liver traumaMethods: This cross-sectional observational study using non-probability convenient sampling technique was done at surgical unit of Liaquat University of Medical and Health Sciences, Jamshoro, for 06 months. After ethical approval from Institute’s Institutional Review Board (IRB), patients presenting to surgical emergency of the hospital between ages 16 to 60 years having blunt or penetrating liver trauma within 04 hours of incident, either road traffic accident, sustaining a fall, sporting injury, knife or stab wound were include while patients of liver trauma conservatively managed or had severe co-morbid, not fit for anesthesia, with multiple organs lesions (polytrauma) and all hepatic injury patients that were hemo-dynamically stable were excluded. SPSS version 23 was used for data analysis keeping p-value <0.05 as significant.Results: Among 136 patients with mean age 32.33±11.23 years, 120(88.2%) were males. 122(89.7%) of the patients were admitted due to liver trauma of blunt variety while 14(10%) with penetrating liver injury. Overall mean duration of hospital stay was 13.1±4.58 days. 41(30%) patients reported intra-abdominal sepsis, followed by recurrent hemorrhage in 33(24%) of patients while in 22(16%) of patients, biliary leakage was observed. An insignificant difference persisted in either surgical intervention in terms of the complication rates.Conclusions: Higher complication rates were observed in patients with peri-hepatic packing, however outcome of both surgical techniques in terms of complication rates were found to be insignificant. Further studies are needed to shed light upon the findings or this study

EVALUATION OF PREHOSPITAL BLOOD PRODUCTS TO ATTENUATE ACUTE COAGULOPATHY OF TRAUMA IN A MODEL OF SEVERE INJURY AND SHOCK IN ANESTHETIZED PIGS

This material is licensed under the terms of the Open Government Licence except where otherwise statedUK Ministry of Defence

T-helper cell polarisation following severe polytrauma

Introduction Severe polytrauma induces an immunosuppressive response and is associated with a very high incidence of nosocomial infections. Previous studies have inferred that this detrimental immune response results from polarisation of the T helper (Th) response towards an anti-inflammatory, TH2 dominated, response at the expense of a bactericidal, Th1 response [1]. Objectives 1) To define alterations in TH cell subsets following severe blunt polytrauma. Methods Patients presenting to the emergency department within 2 hours of severe polytrauma were eligible if intubated either at the scene or in ED. Isolated head injuries and those not expected to survive 24 hours were excluded. EDTA anti-coagulated blood was drawn at 0hr (within 2 hours of injury), at 24 and 72hrs. Samples were immediately lysed, washed, stained and analysed using a standardised human 8-colour TH 1, 2 & 17 panel [2] on an LSR II flow cytometer. A paired white cell count differential was obtained at each sampling point. Patients were followed until discharge or death. Data were analysed using non-parametric statistics, with results presented as median and IQR. Results 15 consecutive severe polytrauma patients requiring Intensive Care Unit (ICU) admission were recruited. Demographic and clinical data are outlined in Figure 1. Twelve (80%) lymphocytosis (3.3x109/L, 2.5 - 4.4x109/L) (Figyre 2A). At 72 hours leukocytes had fallen (P < 0.01, figure 2A) such that 6 (54%) of those surviving were lymphopenic (0.9x109/L, 0.6 - 1.2x109/L). Circulating CD4+ (P = 0.01; Figure 2B) and CD4+CD25+ (P < 0.05) lymphocytes increased over 72 hours. When expressed as a percentage of total circulating lymphocytes no significant change in the proportions of the TH 1, 2 & 17 subpopulations was detected (Figure 2C-E). Conclusions Severe polytrauma patients swiftly become lymphopenic. Although a failure to normalise this during the ICU stay correlates with higher mortality [3] our study of TH cell subtypes demonstrates no evidence of a switch to a detrimental anti-inflammatory TH2 subtype at the expense of the potentially protective bactericidal TH1 subtype