Using Next-Generation Sequencing Transcriptomics To Determine Markers of Post-traumatic Symptoms: Preliminary Findings from a Post-deployment Cohort of Soldiers.

Post-traumatic stress disorder is a concerning psychobehavioral disorder thought to emerge from the complex interaction between genetic and environmental factors. For soldiers exposed to combat, the risk of developing this disorder is twofold and diagnosis is often late, when much sequela has set in. To be able to identify and diagnose in advance those at "risk" of developing post-traumatic stress disorder, would greatly taper the gap between late sequelae and treatment. Therefore, this study sought to determine whether the transcriptome can be used to track the development of post-traumatic stress disorder in this unique and susceptible cohort of individuals. Gene expression levels in peripheral blood samples from 85 Canadian infantry soldiers (n = 58 participants negative for symptoms of post-traumatic stress disorder and n = 27 participants with symptoms of post-traumatic stress disorder) following return from deployment to Afghanistan were determined using RNA sequencing technology. Count-based gene expression quantification, normalization and differential analysis (with thorough correction for confounders) revealed genes associated to PTSD; LRP8 and GOLM1 These preliminary results provide a proof-of-principle for the diagnostic utility of blood-based gene expression profiles for tracking symptoms of post-traumatic stress disorder in soldiers returning from tour. It is also the first to report transcriptome-wide expression profiles alongside a post-traumatic symptom checklist.

Feasibility and transport of packed red blood cells into Special Forces operational conditions.

BACKGROUND: Transfusing packed red blood cells (PRBCs) into Special Forces may provide a survival advantage from hemorrhage-induced battlefield injuries; however, the effect of the unique operational stressors on RBC integrity is not known. METHODS: Pooled PRBCs (20 U) (7 days old), stored in Golden Hour containers, were exposed to the following simulated operational stressors: High-Altitude Low-Opening parachute descent from 30,000 ft, followed by a simulated soldier presence patrol in a climatic chamber set to 48 °C and 9% humidity for 12 hours (test). Biochemical (pH, lactate, potassium, and adenosine triphosphate) and biomechanical (percent hemolysis, deformability, and morphology) were measured to determine the integrity of PRBCs. RESULTS: The simulated parachute descent significantly raised pH (p = 0.025) and potassium (p = 0.014) levels compared with the control; however, this was not clinically significant. Lactate (mmol/L) and adenosine triphosphate levels (0 µmol/g Hgb) were unaffected (p > 0.05). Potassium and pH levels increased with time but not significantly compared with controls. Lactate levels were unaffected with time.Mechanical agitation of PRBCs from the simulated soldier presence patrol did not significantly affect the biochemical (p ≥ 0.08) or biomechanical (p ≥ 0.33) parameters compared with control.Hemolysis was found to be less than 0.8% at the end of 12 hours. No significant difference in RBC morphology and RBC deformability were noted. CONCLUSION: Carrying PRBCs into the austere Special Forces environment is feasible as biochemical and biomechanical markers of RBC stress remain within published transfusion safety parameters when PRBCs were stored in new cold technology containers for 12 hours at 48°C during a simulated Special Forces operation.

The effect of operational stressors on ibuprofen pharmacokinetics.

PURPOSE: To determine whether two of the major operational stressors associated with military missions in Afghanistan: dry heat and long durations of soldier patrol (SP), alter the pharmacokinetics of ibuprofen. METHODS: Thirteen healthy and physically fit participants (19-32 years) were randomized to a four-arm crossover study, as follows: Arm 4 consisted of a simulated 2.5 h SP on a treadmill set at 4.5 km/h, 2% incline (15-min walk/5-min rest cycle) in a climatic chamber set to 42°C, 9% relative humidity. Arm 3 was similar to arm 4 but at room temperature, and arms 1 and 2 were sham SP to 3 and 4, respectively. For the final 2.5 h, participants remained in a semi-supine position. Each participant orally administered one 400-mg Advil Liqui-Gel® capsule. Blood samples were drawn over time and analyzed for (R)-ibuprofen and (S)-plasma ibuprofen concentrations using UPLC/MS/MS. Concentration-time data were analyzed by compartmental methods. RESULTS: Exercise significantly decreased the t(1/2abs) (h) of (S)-ibuprofen (0.26 to 0.17; p = 0.015) and T(max) (h) for both (R)-ibuprofen (0.97 to 0.73; p = 0.008) and (S)-ibuprofen (1.13 to 0.84; p = 0.005). Values for t(lag) (h) also decreased with exercise for both (R)-ibuprofen (0.38 to 0.22; p = 0.005), and (S)-ibuprofen (0.39 to 0.23; p = 0.001). CONCLUSIONS: Exercise stress had a significant impact on the absorption profile of (R)- and (S)-ibuprofen. Excessive self-administration rate and dose may not be due to the military operational stressors of heat and soldier presence patrol.

Physiological strain of next generation combat uniforms with chemical and biological protection: importance of clothing vents.

This study examined whether vents in the arms, legs and chest of new protective assault uniforms (PTAU) reduced heat strain at 35 °C during a low dressed state (DSlow), and subsequently improved tolerance time (TT) after transitioning to DShigh compared with the battle dress uniform and overgarment (BDU+O). Small but significant reductions in rectal temperature (Tre), heart rate and vapour pressures over the thigh and shin were observed during DSlow with vents open (37.9 ± 0.2 °C, 120 ± 10 b/min, 3.7 ± 0.4 and 3.5 ± 1.0 kPa) versus closed (38.0 ± 0.1 °C, 127 ± 5 b/min, 4.3 ± 0.3 and 4.6 ± 0.5 kPa). During DShigh Tre was reduced and TT increased significantly with the PTAUs (1.1 ± 0.2 °C/h and 46 ± 24 min) versus BDU+O (1.6 ± 0.2 °C/h and 33 ± 16 min). The vents marginally reduced heat strain during DSlow and extended TT during DShigh) compared with BDU+O. Practitioner Summary: Clothing vents in chemical and biological protective uniforms can assist with heat transfer in situations where the uniforms must be worn for extended periods prior to exposure to a hazardous condition. Once the vents are closed, exposure time is increased and the increase in body temperature reduced.

Early gene expression profiles during intraoperative myocardial ischemia-reperfusion in cardiac surgery.

OBJECTIVE: The effects of cold cardioplegic arrest and reperfusion on human ventricular gene expression are unknown. We tested the hypothesis that intraoperative ischemia-reperfusion under conditions of blood cardioplegic arrest would induce a unique myocardial genomic profile indicative of a cardioprotective response. METHODS: Right ventricular samples were serially acquired during surgical repair of ventricular septal defect. RESULTS: Expression profiling revealed 3 patterns of gene expression: (1) increased expression above control levels within 1 hour of cardioplegic arrest, with further amplification during early reperfusion; (2) increased expression limited to the reperfusion phase; and (3) reduced expression during reperfusion. Functional annotation and network mapping of differentially expressed genes indicated activation of multiple signaling pathways regulated by phosphatidylinositide 3'-OH kinase convergent on cellular growth and reparative programs. Also observed was increased expression of genes regulating hemoglobin synthesis, suggesting a novel cardioprotective pathway evoked during ischemia-reperfusion. CONCLUSION: Reversible myocardial ischemia-reperfusion during cardiac surgery is associated with an immediate genomic response that predicts a net cardioprotective phenotype.

Age-related differences in myocardial hydrogen ion buffering during ischemia.

BACKGROUND: During myocardial ischemia, accumulation of end products from anaerobic glycolysis (hydrogen ions (H(+)), lactate) can cause cellular injury, consequently affecting organ function. The cells' ability to buffer H(+) (buffering capacity (BC)) plays an important role in ischemic tolerance. Age related differences in myocardial lactate and H(+) accumulation (one hour of ischemia) as well as differences in BC, myoglobin (Mb) and histidine (His) contents in the left (LV) and right (RV) ventricles were assessed in neonatal compared to adult pigs. The BC of the septum was also compared. METHODS AND RESULTS: Neonatal RV and LV had lactate accumulations of 43% and 63% and significantly greater H(+) (p < 0.004) compared to the adult. In the neonate LV, BC was 17% significantly poorer (p = 0.0001), had 33% lower Mb (p = 0.0002) and 15% lower His content (p = 0.0004) when compared to the adult. In the RV, despite similar BC between the neonate and adult, myoglobin content was 36% (p = 0.0004) lower in the neonate. The neonate septum had a BC that was 11% lower than that of the adult. With maturation, the adult LV had a BC that was 10% greater (p < 0.01) than the RV while the septum mirrored that of the LV. CONCLUSIONS: During maturation to adulthood, the BC of the septum begins to closely resemble the LV. Neonatal hearts have a potentially greater vulnerability to acid-base disturbances during ischemia in both ventricles when compared to hearts of adults. This is due to lower levels of myoglobin and histidine in the young, which could render them more susceptible to injury during ischemia. During myocardial ischemia, H(+) and lactate accumulation may pose deleterious effects on the heart. The ability to buffer H(+) (buffering capacity, BC) affects ischemic tolerance. Although lactate accumulation during 1 h of global ischemia was similar between ventricles of neonatal and adult swine, H(+) accumulation was greater and BC, Mb and His content were lower. With maturation, LV BC was higher than the RV while septum developmentally resembled the LV. Thus, hearts of neonates may be at a greater risk of ischemic injury compared to hearts of adults.

The remote ischemic preconditioning stimulus modifies gene expression in mouse myocardium.

BACKGROUND: We have recently demonstrated that remote ischemic preconditioning reduces ischemia-reperfusion injury in animal models. The mechanisms by which the remote ischemic preconditioning stimulus exerts its effect remain to be fully defined, and its effect on myocardial gene expression is unknown. We tested the hypothesis that remote ischemic preconditioning modifies myocardial gene expression immediately after the remote ischemic preconditioning stimulus (early phase) and 24 hours later (late phase). METHODS: Twenty male (C57BL/6) 10- to 12-week-old mice were randomized into 4 groups: group 1 (control, early phase; n = 5), group 2 (remote ischemic preconditioning, early phase; n = 5), group 3 (control, late phase; n = 5), and group 4 (remote ischemic preconditioning, late phase; n = 5). Groups 2 and 4 underwent remote ischemic preconditioning induced by 6 cycles of 4 minutes of occlusion and 4 minutes of reperfusion of the femoral artery. Groups 1 and 2 were killed 15 minutes after completion of sham procedure or remote ischemic preconditioning, and the hearts were removed and frozen in liquid nitrogen. Groups 3 and 4 were killed 24 hours after remote ischemic preconditioning, and the hearts were harvested in the same fashion. Gene expression was assessed by using the Affymetrix MG-430A chip (Affymetrix, Santa Clara, Calif). RESULTS: Data filtering (P < .05, analysis of variance) and hierarchic 2-way clustering identified significant differences in gene expression among the 4 groups. Genes involved in protection against oxidative stress (eg, Hadhsc, Prdx4, and Fabp4) and cytoprotection (Hsp73) were upregulated, whereas many proinflammatory genes (eg, Egr-1 and Dusp 1 and 6) were suppressed. CONCLUSION: A simple remote ischemic preconditioning stimulus modifies myocardial gene expression by upregulating cardioprotective genes and suppressing genes potentially involved in the pathogenesis of ischemia-reperfusion injury.

Cardioprotective stress response in the human fetal heart.

OBJECTIVE: We propose that the fetal heart is highly resilient to hypoxic stress. Our objective was to elucidate the human fetal gene expression profile in response to simulated ischemia and reperfusion to identify molecular targets that account for the innate cardioprotection exhibited by the fetal phenotype. METHODS: Primary cultures of human fetal cardiac myocytes (gestational age, 15-20 weeks) were exposed to simulated ischemia and reperfusion in vitro by using a simulated ischemic buffer under anoxic conditions. Total RNA from treated and baseline cells were isolated, reverse transcribed, and labeled with Cy3 or Cy5 and hybridized to a human cDNA microarray for expression analysis. This analysis revealed a highly significant (false discovery rate, <3%) suppression of interleukin 6 transcript levels during the reperfusion phase confirmed by means of quantitative polymerase chain reaction (0.25 +/- 0.11-fold). Interleukin 6 signaling during ischemia and reperfusion was assessed at the protein expression level by means of Western measurements of interleukin 6 receptor, the signaling subunit of the interleukin 6 receptor complex (gp130), and signal transducer of activated transcription 3. Posttranslational changes in the protein kinase B signaling pathway were determined on the basis of the phosphorylation status of protein kinase B, mitogen-activated protein kinase, and glycogen synthase kinase 3beta. The effect of suppression of a prohypertrophic kinase, integrin-linked kinase, with short-interfering RNA was determined in an ischemia and reperfusion-stressed neonatal rat cardiac myocyte model. Endogenous secretion of interleukin 6 protein in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS: Human fetal cardiac myocytes exhibited a significantly lower rate of apoptosis induction during ischemia and reperfusion and after exposure to staurosporine and recombinant interleukin 6 compared with that observed in neonatal rat cardiac myocytes ( P < .05 for all comparisons, analysis of variance). Exposure to exogenously added recombinant interleukin 6 increased the apoptotic rate in both rat and human fetal cardiac myocytes ( P < .05). Short-interfering RNA-mediated suppression of integrin-linked kinase, a prohypertrophy upstream kinase regulating protein kinase B and glycogen synthase kinase 3beta phosphorylation, was cytoprotective against ischemia and reperfusion-induced apoptosis in neonatal rat cardiac myocytes ( P < .05). CONCLUSIONS: Human fetal cardiac myocytes exhibit a uniquely adaptive transcriptional response to ischemia and reperfusion that is associated with an apoptosis-resistant phenotype. The stress-inducible fetal cardiac myocyte gene repertoire is a useful platform for identification of targets relevant to the mitigation of cardiac ischemic injury and highlights a novel avenue involving interleukin 6 modulation for preventing the cardiac myocyte injury associated with ischemia and reperfusion.

Gene expression profiles in children undergoing cardiac surgery for right heart obstructive lesions.

BACKGROUND: The global myocardial stress response during cardiac surgery has not been systematically studied, nor is it known whether the response of the neonatal myocardium is intrinsically different from that of older children. To determine the age-related molecular basis of this response, we conducted microarray-based differential gene expression profiling on right ventricular tissue samples acquired in patients of varying ages with right ventricular outflow tract obstruction. METHODS: We studied gene expression profiles in 24 patients during operations for lesions involving right ventricular outflow tract obstruction age stratified into group I (7 patients, aged 5 to 66 days; mean, 30 days) and group II (17 patients, aged 4 months to 12.5 years; mean, 2.8 years). Myocardial samples were taken from the right ventricular outflow tract after aortic occlusion and archived in liquid nitrogen. RNA isolation, fluorescence labeling of complementary DNA, hybridization to spotted arrays containing 19,008 characterized or unknown human complementary DNAs, and quantitative fluorescence scanning of gene-expression intensity were performed at the University of Toronto Health Network Microarray Centre. Data were analyzed with the Significance Analysis for Microarrays program. Minimum Information About Microarray Experiments-compliant, log2-normalized data sets were compared to ascertain potential statistical differences in gene expression between patient groups. RESULTS: There were no hospital deaths or major postoperative morbid events. We identified 50 transcripts differentially expressed in the neonatal group (the predicted false discovery rate was <0.8 transcripts). The neonatal pattern of gene expression (group I) was dominated by genes with literature-validated cardioprotective, antihypertrophic, and antiproliferative properties, including increases in atrial natriuretic peptide, protein phosphatase 2A, small GTPase rap1, and protein inhibitor of activated STAT protein, PIASy. Several transcripts have not been previously reported in heart. CONCLUSIONS: Neonatal myocardium has a unique pattern of gene expression, which may result from developmental (age-related) differences or reflect a more severe disease phenotype independent of age effects per se. The neonatal transcript profile seems to reflect a stress-induced protective program composed of genes with functions diametrically opposed to those expected to be related to the pathogenesis of critical right ventricular outflow tract obstruction, thus revealing a novel and compensatory antidisease transcriptional response in the neonatal heart.