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Background: Respiratory failure can be a severe complication after polytrauma. Extensive systemic inflammation due to surgical interventions, as well as exacerbated post-traumatic immune responses influence the occurrence and progression of respiratory failure. This study investigated the effect of different surgical treatment modalities as well as combined inhibition of the complement component C5 and the toll-like receptor molecule CD14 (C5/CD14 inhibition) on the pulmonary microRNA (miRNA) signature after polytrauma, using a translational porcine polytrauma model., Methods: After induction of general anesthesia, animals were subjected to polytrauma, consisting of blunt chest trauma, bilateral femur fractures, hemorrhagic shock, and liver laceration. One sham group (n=6) and three treatment groups were defined; Early Total Care (ETC, n=8), Damage Control Orthopedics (DCO, n=8), and ETC + C5/CD14 inhibition (n=4). Animals were medically and operatively stabilized, and treated in an ICU setting for 72 h. Lung tissue was sampled, miRNAs were isolated, transcribed, and pooled for qPCR array analyses, followed by validation in the individual animal population. Lastly, mRNA target prediction was performed followed by functional enrichment analyses., Results: The miRNA arrays identified six significantly deregulated miRNAs in lung tissue. In the DCO group, miR-129, miR-192, miR-194, miR-382, and miR-503 were significantly upregulated compared to the ETC group. The miRNA expression profiles in the ETC + C5/CD14 inhibition group approximated those of the DCO group. Bioinformatic analysis revealed mRNA targets and signaling pathways related to alveolar edema, pulmonary fibrosis, inflammation response, and leukocytes recruitment. Collectively, the DCO group, as well as the ETC + C5/CD14 inhibition group, revealed more anti-inflammatory and regenerative miRNA expression profiles., Conclusion: This study showed that reduced surgical invasiveness and combining ETC with C5/CD14 inhibition can contribute to the reduction of pulmonary complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zhou, Groven, Horst, Mert, Greven, Mollnes, Huber-Lang, van Griensven, Hildebrand and Balmayor.)

Introduction: Two trauma treatment principles are Early Total Care (ETC), and Damage Control Orthopedics (DCO). Cellular mechanisms that underlie the connection between treatment type, its systemic effects, and tissue regeneration are not fully known. Therefore, this study aimed to: 1) profile microRNA (miRNA) expression in plasma derived Extracellular Vesicles (EVs) from a porcine multiple trauma model at different timepoints, comparing two surgical treatments; and 2) determine and validate the miRNA's messengerRNA (mRNA) targets., Methods: The porcine multiple trauma model consisted of blunt chest trauma, liver laceration, bilateral femur fractures, and controlled haemorrhagic shock. Two treatment groups were defined, ETC (n=8), and DCO (n=8). Animals were monitored under Intensive Care Unit-standards, blood was sampled at 1.5, 2.5, 24, and 72 hours after trauma, and EVs were harvested from plasma. MiRNAs were analysed using quantitative Polymerase Chain Reaction arrays. MRNA targets were identified in silico and validated in vivo in lung and liver tissue., Results: The arrays showed distinct treatment specific miRNA expression patterns throughout all timepoints, and miRNAs related to the multiple trauma and its individual injuries. EV-packed miRNA expression in the ETC group was more pro-inflammatory, indicating potentially decreased tissue regenerative capacities in the acute post-traumatic phase. In silico target prediction revealed several overlapping mRNA targets among the identified miRNAs, related to inflammation, (pulmonary) fibrosis, and Wnt-signalling. These were, among others, A Disintegrin and Metalloproteinase domain-containing protein 10, Collagen Type 1 Alpha 1 Chain, Catenin Beta Interacting Protein 1, and Signal Transducers and Activators of Transcription 3. Validation of these mRNA targets in the lung showed significant, treatment specific deregulations which matched the expression of their upstream miRNAs. No significant mRNA deregulations were observed in the liver., Discussion: This study showed treatment specific, EV-packed miRNA expression patterns after trauma that correlated with mRNA expressions in the lungs, target organs over distance. A systemic response to the increased surgical trauma in the ETC group was identified, with various miRNAs associated with injuries from the trauma model, and involved in (systemic) inflammation, tissue regeneration. EV-transported miRNAs demonstrated a clear role in multiple trauma, warranting further research into tissue-tissue talk and therapeutic applications of EVs after trauma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Groven, Greven, Mert, Horst, Zhao, Blokhuis, Huber-Lang, Hildebrand and van Griensven.)

Introduction: Patients after polytrauma suffer from posttraumatic immune system dysregulation and multiple organ dysfunction. Genome-wide microarray profiling in monocytes revealed a regulatory network of inflammatory markers around the transcription factor AP-1 in severely injured patients. Recent research focuses on the role of neutrophils in posttraumatic inflammation. The aim of this study was, therefore, to evaluate the impact of this inflammatory network in neutrophils., Materials and Methods: Blood sampling and neutrophil separation were performed on admission of the patient and at 6 h, 12 h, 24 h, 48 h, and 72 h after trauma. Neutrophil expression levels of the target genes c-Jun, c-Fos, BCL2A, MMP-9, TIMP-1, ETS-2, IL-1β, and MIP-1β were quantified by RT-qPCR. Patients were assorted into groups according to distinct clinical parameters like massive transfusion (>10 RBC units/24 h), injury severity (ISS), 90-d survival, and the presence of traumatic brain injury (defined by ICI on head CT). Statistics were calculated by Mann-Whitney Rank-Sum Test, Receiver Operating Curves, and binary multiple logistic regression., Results: Forty severely injured patients (mean ISS 36 ± 14) were included. BCL2A, MMP-9, TIMP-1, and ETS2 levels showed a significant correlation to 90-d-survival in the early posttraumatic period (6 h-24 h). Furthermore, differential BCL2A, IL-1β, MIP-1β, and MMP-9 regulation was observed in patients requiring massive transfusion. We could further show a significant TIMP-1 response in trauma PMN associated with traumatic brain injury., Conclusions: This study of seriously injured patients highlights very early posttraumatic transcriptional changes in PMNs, which were clearly associated with posttraumatic events and outcomes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In the stabilization of femoral fractures in mono- and polytrauma, clinical practice has shown better care through intramedullary nailing. However, the reason why this is the case is not fully understood. In addition to concomitant injuries, the immunological aspect is increasingly coming to the fore. Neutrophil granulocytes (PMNL), in particular next to other immunological cell types, seem to be associated with the fracture healing processes. For this reason, the early phase after fracture (up to 72 h after trauma) near the fracture zone in muscle tissue was investigated in a pig model., Material and Methods: A mono- and polytrauma pig model (sole femur fracture or blunt thoracic trauma, hemorrhagic shock, liver laceration, and femur fracture) was used to demonstrate the immunological situation through muscle biopsies and their analysis by histology and qRT-PCR during a 72 h follow-up phase. Two stabilization methods were used (intramedullary nail vs. external fixator) and compared with a nontraumatized sham group., Results: Monotrauma shows higher PMNL numbers in muscle tissue compared with polytrauma (15.52 ± 5.39 mono vs. 8.23 ± 3.36 poly; p = 0.013), regardless of the treatment strategy. In contrast, polytrauma shows a longer lasting invasion of PMNL (24 h vs. 72 h). At 24 h in the case of monotrauma, the fracture treated with external fixation shows more PMNL than the fracture treated with intramedullary nailing (p = 0.026). This difference cannot be determined in polytrauma probably caused by a generalized immune response. Both monotrauma and polytrauma show a delayed PMNL increase in the muscle tissue of the uninjured side. The use of intramedullary nailing in monotrauma resulted in a significant increase in IL-6 (2 h after trauma) and IL-8 (24 and 48 h after trauma) transcription., Conclusion: The reduction of PMNL invasion into the nearby muscle tissue of a monotrauma femur fracture stabilized by intramedullary nailing supports the advantages found in everyday clinical practice and therefore underlines the usage of nailing. For the polytrauma situation, the fixation seems to play a minor role, possibly due to a generalized immune reaction.

Background: Predictive biomarkers in biofluids are the most commonly used diagnostic method, but established markers in trauma diagnostics lack accuracy. This study investigates promising microRNAs (miRNA) released from affected tissue after severe trauma that have predictive values for the effects of the injury., Methods: A retrospective analysis of prospectively collected data and blood samples of n = 33 trauma patients (ISS ≥ 16) is provided. Levels of miR-9-5p, -124-3p, -142-3p, -219a-5p, -338-3p and -423-3p in severely injured patients (PT) without traumatic brain injury (TBI) or with severe TBI (PT + TBI) and patients with isolated TBI (isTBI) were measured within 6 h after trauma., Results: The highest miR-423-3p expression was detected in patients with severe isTBI, followed by patients with PT + TBI, and lowest levels were found in PT patients without TBI (2 -∆∆Ct , p = 0.009). A positive correlation between miR-423-3p level and increasing AIS head ( p = 0.001) and risk of mortality (RISC II, p = 0.062) in trauma patients ( n = 33) was found. ROC analysis of miR-423-3p levels revealed them as statistically significant to predict the severity of brain injury in trauma patients ( p = 0.006). miR-124-3p was only found in patients with severe TBI, miR-338-3p was shown in all trauma groups. miR-9-5p, miR-142-3p and miR-219a-5p could not be detected in any of the four groups., Conclusion: miR-423-3p expression is significantly elevated after isolated traumatic brain injury and predictable for severe TBI in the first hours after trauma. miR-423-3p could represent a promising new biomarker to identify severe isolated TBI.

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

Background: A high percentage of the critically ill polytrauma patients develop acute kidney injury (AKI) secondary to trauma and are therefore prone to high morbidity and mortality rates. One of the main objectives in these cases is the fast detection of the condition and continuous rigorous monitoring of the patients. Currently the panel of biomarkers available for monitoring and for the prognosis of AKI is limited. Numerous studies have proven the importance of microRNAs in this field. In this actualization paper we wish to summarize the most relevant microRNAs that can be used as biomarkers for patients with AKI., Methods: For this paper, we looked into the studies available in scientific databases such as PubMed and Scopus. For the analysis we used the following key words: "miRNAs biomarker", "acute kidney injury AKI", "genetic expression in AKI", and "epigenetic microRNAs biomarkers in AKI"., Results: Numerous studies have shown high specificity for certain microRNA species in the case of patients with AKI. Moreover, they have reported a series of microRNAs that present high specificity and that have a strong expression in fluids that can be sampled through non-invasive methods, such as urine and saliva., Conclusions: The expression of microRNAs can be successfully used in the future as a non-invasive method for the evaluation and monitoring of AKI patients.

Background: Valproic acid (VPA) is a histone deacetylase inhibitor that improves outcomes in large animal models of trauma. However, its protective mechanism of action is not completely understood. We sought to characterize the genetic changes induced by VPA treatment following traumatic injuries., Methods: Six female Yorkshire swine were subjected to traumatic brain injury (controlled cortical impact), polytrauma (liver and splenic laceration, rib fracture, rectus crush), and hemorrhagic shock (HS, 40% total blood volume). Following 2 hours of HS, animals were randomized to resuscitation with normal saline (NS) or NS + 150 mg/kg of intravenous VPA (n = 3/cohort, 18 samples total). Blood samples were collected for isolation of peripheral blood mononuclear cells at three distinct time points: baseline, 6 hours following injuries, and on postinjury day 1. RNA was extracted from peripheral blood mononuclear cells and sequenced. Differential expression analysis (false discovery rate < 0.001 and p value <0.001) and gene set enrichment (Panther Gene Ontology and Ingenuity Pathway Analysis) was used to compare VPA to non-VPA-treated animals., Results: A total of 628 differentially expressed RNA transcripts were identified, 412 of which were used for analysis. There was no difference between treatment groups at baseline. The VPA-induced genetic changes were similar at 6 hours and on postinjury day 1. Upregulated genes were associated with gene expression (p 2.13E-34), cellular development (1.19E-33), cellular growth and proliferation (1.25E-30), and glucocorticoid receptor signaling (8.6E-21). Downregulated genes were associated with cell cycle checkpoint regulation (3.64E-22), apoptosis signaling (6.54E-21), acute phase response signaling (5.84E-23), and the inflammasome pathway (1.7E-19)., Conclusion: In injured swine, VPA increases the expression of genes associated with cell survival, proliferation, and differentiation and decreases those associated with cell death and inflammation. These genetic changes could explain the superior clinical outcomes in VPA-treated animals, including smaller brain lesion size and improved neurologic recovery.

Background: Cell free DNA (cfDNA) was recently suggested as a new marker of sepsis and poor outcome in ICU patients. Procalcitonin has also been the focus of attention as an early marker for systemic inflammation and sepsis., Methods: cfDNA, procalcitonin (PCT), C-reactive protein (CRP), and lactate levels were measured in 30 ICU patients with multiple trauma or after major surgery on the first day of admission and on 5th and 7th days for PCT, CRP, and lactate. cfDNA was measured by real-time PCR, PCT by ELISA, CRP immunoturbidimetrically, and lactate spectrophotometrically. SOFA score and Injury Severity Score (ISS) for trauma patients were calculated., Results: Significantly higher levels of cfDNA were observed in non-survivor patients in comparison to survivors and in patients with sepsis in comparison to those without sepsis (p = 0.002 and p = 0.02, respectively). The ROC curve was calculated for cfDNA as a predictor of outcome, the area under the curve (AUC) was 0.847 (95% CI: 0.669 - 0.952), at a cutoff value of 15500 ng/µL, sensitivity = 83.3%, specificity = 77.8% (p < 0.0001). As a prognostic marker of sepsis, the AUC for cfDNA was 0.788 (95% CI: 0.601 - 0.915), sensitivity = 56.25%, specificity = 100% (p = 0.0007). Day 5 PCT levels significantly correlated with SOFA scores on day 5, ISS on admission (p < 0.001 and p = 0.028, respectively), and a significant elevation of its levels was observed in non-survivor patients compared to survivors (p = 0.001). As a predictor of sepsis, PCT showed a sensitivity of 81.3%, specificity of 100% on day 5, (AUC: 0.987, 95% CI: 0.955 - 1.00); at a cutoff value of 202.90 pg/mL (p = 0.001). As a predictor of outcome, PCT on day 5 showed a sensitivity of 94.0% and a specificity of 78.0% at a cutoff value of 194.40 pg/mL (p = 0.001). Day 1 CRP correlated with ISS on admission, and on day 5 it correlated with SOFA score 5, while lactate correlated with length of stay on days 1, 5, and 7, and its levels were significantly higher in non-survivors on days 5 and 7., Conclusions: cfDNA is a good predictor of patient outcome in ICU and to a lesser extent as a marker of sepsis. PCT is another promising marker that can complement cfDNA to reach better patient management. Other markers can help in less severe cases.

Background: One of the most severe conditions specific to the critically ill polytrauma patient is traumatic brain injury and traumatic spinal cord injury. The mortality rate is high in the case of these patients, both because of the direct traumatic lesions, and because of the pathophysiological imbalances associated with trauma. Amongst the most common pathologies associated with the critically ill polytrauma patients responsible for a lower survival rate, are redox imbalance, systemic inflammatory response, infections, and multiple organ dysfunction syndrome., Methods: For this study, was analysed the literature available on PubMed. The key words used in the search were "traumatic brain injury", "spinal cord injury", "microRNAs expression", "polytrauma patients", and "biomarkers"., Results: For the study were selected 34 science articles. The oxidative attack on lipids is responsible for the biosynthesis of an increased quantity of free radicals, which further intensifies and aggravates the redox status in these patients., Conclusions: A new era for biomarkers is represented by the expression of miRNAs. In the case of the critically ill polytrauma patient, using miRNAs' expression as biomarkers for the evaluation and monitoring of the molecular and pathophysiological dysfunctions can bring a range of valuable answers that could contribute to an increased survival rate.

Critical polytrauma patients present a series of pathophysiological disturbances, biochemical and molecular dysfunction, which comprise to be the major cause of intensive care unit admission. In regard to molecular damage, there exists a series of factors, which all together contribute to the aggravation of the clinical status leading to increased mortality rate in these patients. One of the most important biochemical factors involved is the nuclear transcription factor B (NF-κB). Impaired NF-κB functioning is reflected on the clinical status of the patient through increased production of pro-inflammatory molecule, leading to multiple organ dysfunction syndrome. In addition to this, through microRNAs interactions, various pathophysiological as well as biochemical disturbances are produced, which altogether further reduce the patient's survival rate. In this paper, we would like to present the modifications seen in the expression of NF-κB in critically polytraumatized patients with sepsis. In additions to this, we would like to discuss the correlation between the microRNAs and its further implications in clinical status of these patients.

Background: The complexity of the cases of critically ill polytrauma patients is given by both the primary, as well as the secondary, post-traumatic injuries. The severe injuries of organ systems, the major biochemical and physiological disequilibrium, and the molecular chaos lead to a high rate of morbidity and mortality in this type of patient. The 'gold goal' in the intensive therapy of such patients resides in the continuous evaluation and monitoring of their clinical status. Moreover, optimizing the therapy based on the expression of certain biomarkers with high specificity and sensitivity is extremely important because of the clinical course of the critically ill polytrauma patient. In this paper we wish to summarize the recent studies of biomarkers useful for the intensive care unit (ICU) physician., Methods: For this study the available literature on specific databases such as PubMed and Scopus was thoroughly analyzed. Each article was carefully reviewed and useful information for this study extracted. The keywords used to select the relevant articles were "sepsis biomarker", "traumatic brain injury biomarker" "spinal cord injury biomarker", "inflammation biomarker", "microRNAs biomarker", "trauma biomarker", and "critically ill patients"., Results: For this study to be carried out 556 original type articles were analyzed, as well as case reports and reviews. For this review, 89 articles with relevant topics for the present paper were selected. The critically ill polytrauma patient, because of the clinical complexity the case presents with, needs a series of evaluations and specific monitoring. Recent studies show a series of either tissue-specific or circulating biomarkers that are useful in the clinical status evaluation of these patients., Conclusions: The biomarkers existing today, with regard to the critically ill polytrauma patient, can bring a significant contribution to increasing the survival rate, by adapting the therapy according to their expressions. Nevertheless, the necessity remains to research new non-invasive diagnostic methods that present with higher specificity and selectivity.

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.

Objective: The objective of this study was to conduct a systematic survey of common precursor microRNA (pre-miRNA) single nucleotide polymorphisms (SNPs) and evaluate their clinical relevance in patients with major blunt trauma., Background: Recent evidence indicates that small noncoding RNA molecules known as miRNAs can function as important negative gene regulators and are implicated in the pathogenesis of various diseases., Methods: We conducted a 2-stage study to examine the impact of 9 selected SNPs with potential functional significance on the susceptibility to sepsis of 1268 trauma patients (1 screening cohort, n = 666) and 2 independent validated cohorts (n = 286 and n = 316, respectively) in China., Results: Among the 9 selected SNPs with potential functional significance, only 1 (miR-608 rs4919510) was found to be strongly associated with a higher risk of developing sepsis and multiple organ dysfunction in all 3 independent study cohorts. An even stronger association was observed for the rs4919510 polymorphism when combining these 3 study cohorts together. In addition, the rs4919510 polymorphism showed a significant correlation with a higher production of proinflammatory cytokines and a lower production of anti-inflammatory cytokines. In vitro experiments further indicated that the G→C variant of this polymorphism could significantly increase the expression of mature miR-608., Conclusions: Our results indicate that the rs4919510G/C SNP in hsa-mir-608 may be a prognostic biomarker for sepsis in patients with major trauma. Further characterization of miRNA SNPs may open new avenues for studying sepsis and developing novel therapeutic approaches.

Background: Recruitment of bone marrow derived endothelial progenitor cells (BMDEPCs) alleviates multiple organ injury (MOI) and improves outcomes. However, mechanisms mediating BMDEPC recruitment following septic MOI remain largely unknown. This study characterized the kinetics of BMDEPC recruitment and proliferation and defined the role of NF-κB in regulating BMDEPC recruitment and proliferation., Methods and Main Findings: Chimeric mice with an intact or disrupted NF-κB p50 gene and BMDEPC-restricted expression of green fluorescent protein were created and injected with LPS (2 mg/kg, i.p.). BMDEPC recruitment and proliferation in multiple organs were quantified. BMDEPC recruitment and proliferation are highly organ-dependent. Lungs had the highest number of BMDEPC recruitment, whereas heart, liver and kidney had only a small fraction of the number of BMDEPCs in lungs. Number of proliferating BMDEPCs was several-fold higher in lungs than in other 3 organs. Kinetically, BMDEPC recruitment into different organs showed different time course profiles. NF-κB plays obligatory roles in mediating BMDEPC recruitment and proliferation. Universal deletion of NF-κB p50 gene inhibited LPS-induced BMDEPC recruitment and proliferation by 95% and 69% in heart. However, the contribution of NF-κB to these regulations varies significantly between organs. In liver, universal p50 gene deletion reduced LPS-induced BMDEPC recruitment and proliferation only by 49% and 35%. NF-κB activities in different tissue compartments play distinct roles. Selective p50 gene deletion either in stromal/parenchymal cells or in BM/blood cells inhibited BMDEPC recruitment by a similar extent. However, selective p50 gene deletion in BM/blood cells inhibited, but in stromal/parenchymal cells augmented BMDEPC proliferation., Conclusions: BMDEPC recruitment and proliferation display different kinetics in different organs following endotoxemic MOI. NF-κB plays obligatory and organ-dependent roles in regulating BMDEPC recruitment and proliferation. NF-κB activities in different tissue compartments play distinct roles in regulating BMDEPC proliferation.

Hemorrhagic shock is a leading cause of trauma-related mortality in both civilian and military settings. Resuscitation often results in reperfusion injury and survivors are susceptible to developing multiple organ failure (MOF). The impact of fed state on the overall response to shock and resuscitation has been explored in some murine models but few clinically relevant large animal models. We have previously used metabolomics to establish that the fed state results in a different metabolic response in the porcine liver following hemorrhagic shock and resuscitation. In this study, we used our clinically relevant model of hemorrhagic shock and polytrauma and the Illumina HiSeq platform to determine if the liver transcriptomic response is also altered with respect to fed state. Functional analysis of the response to shock and resuscitation confirmed several typical responses including carbohydrate metabolism, cytokine inflammation, decreased cholesterol synthesis, and apoptosis. Our findings also suggest that the fasting state, relative to a carbohydrate prefed state, displays decreased carbohydrate metabolism, increased cytoskeleton reorganization and decreased inflammation in response to hemorrhagic shock and reperfusion. Evidence suggests that this is a consequence of a shrunken, catabolic state of the liver cells which provides an anti-inflammatory condition that partially mitigates hepatocellar damage.

In polytrauma, injuries that may be surgically treated under regular circumstances due to a systemic inflammatory response become life-threatening. The inflammatory response involves a complex pattern of humoral and cellular responses and the expression of related factors is thought to be governed by genetic variations. This aim of this paper is to examine the influence of interleukin (IL) 6 single nucleotide polymorphism (SNP) -174C/G and -596G/A on the treatment outcome in severely injured patients. Forty-seven severely injured patients were included in this study. Patients were assigned an Injury Severity Score. Blood samples were drawn within 24 h after admission (designated day 1) and on subsequent days (24, 48, 72 hours and 7 days) of hospitalization. The IL-6 levels were determined through ELISA technique. Polymorphisms were analyzed by a method of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR). Among subjects with different outcomes, no statistically relevant difference was found with regards to the gene IL-6 SNP-174G/C polymorphism. More than a half of subjects who died had the SNP-174G/C polymorphism, while this polymorphism was represented in a slightly lower number in survivors. The incidence of subjects without polymorphism and those with heterozygous and homozygous gene IL-6 SNP-596G/A polymorphism did not present statistically significant variations between survivors and those who died. The levels of IL-6 over the observation period did not present any statistically relevant difference among subjects without the IL-6 SNP-174 or IL- 6 SNP -596 gene polymorphism and those who had either a heterozygous or a homozygous polymorphism.

The purpose of this study was to identify critical genes associated with septic multiple trauma by comparing peripheral whole blood samples from multiple trauma patients with and without sepsis. A microarray data set was downloaded from the Gene Expression Omnibus (GEO) database. This data set included 70 samples, 36 from multiple trauma patients with sepsis and 34 from multiple trauma patients without sepsis (as a control set). The data were preprocessed, and differentially expressed genes (DEGs) were then screened for using packages of the R language. Functional analysis of DEGs was performed with DAVID. Interaction networks were then established for the most up- and down-regulated genes using HitPredict. Pathway-enrichment analysis was conducted for genes in the networks using WebGestalt. Fifty-eight DEGs were identified. The expression levels of PLAU (down-regulated) and MMP8 (up-regulated) presented the largest fold-changes, and interaction networks were established for these genes. Further analysis revealed that PLAT (plasminogen activator, tissue) and SERPINF2 (serpin peptidase inhibitor, clade F, member 2), which interact with PLAU, play important roles in the pathway of the component and coagulation cascade. We hypothesize that PLAU is a major regulator of the component and coagulation cascade, and down-regulation of PLAU results in dysfunction of the pathway, causing sepsis.

Objectives: Severe sepsis after trauma still associated with a high mortality rate in intensive care units (ICU). In this study we aimed to identify genes related to multiple trauma complicated by severe sepsis., Materials and Methods: The gene expression profile dataset GSE12624 including 36 samples of traumatic patients not complicated by sepsis and 34 traumatic patients complicated by sepsis was downloaded from GEO (Gene Expression Omnibus) database. The limma package in R was applied to identify differentially expressed genes (DEGs) between these two groups of samples. All the DEGs were divided into up- and down-regulation groups according to the changes of their expression value, which were then subjected to GO enrichment analysis. Two genes with largest changes among the up- and down-regulation groups were selected. Interaction networks based on these two genes were constructed using HitPredict software and then pathway enrichment analysis for the networks were performed by WebGestalt software., Results: A total of 21 up-regulated genes and 37 down-regulated genes were obtained, which were mainly related to GO terms "endopeptidase inhibitor activity" and "response to wounding", respectively. The |logFC| of genes PLAU (urokinase-type plasminogen activator) and MMP8 (matrix metalloproteinase-8) ranked first in down-regulated or up-regulated list. There were 18 genes which can interact with PLAU at a high degree of confidence while there were 5 genes with MMP8. Further analysis showed that PLAU was closely associated with the pathway "complement and coagulation cascades"., Conclusions: PLAU and MMP8 may act as potential targets for diagnosis and therapy of trauma complicated by sepsis.

Introduction: The receptor for advanced glycation end products (RAGE) has been considered as one of the major pattern recognition receptors and plays an important role in the development of sepsis and multiple organ dysfunction in critical illnesses. Although genetic variants of the RAGE gene have been shown to be well associated with susceptibility to some inflammatory diseases, little is known about their clinical relevance in the development of sepsis in critical ill patients., Methods: Four genetic variants were selected from the entire RAGE gene and genotyped using pyrosequencing and polymerase chain reaction-length polymorphism methods. Association studies were performed in two independent Chinese Han populations., Results: Among the four genetic variants, only the rs1800625 polymorphism was significantly associated with sepsis morbidity rate and multiple organ dysfunction (MOD) scores in patients with major trauma both in Chongqing (n = 496) and Zhejiang (n = 232) districts, respectively. Results from ex vivo responsiveness of peripheral blood leukocytes indicated that the rs1800625 polymorphism was well associated with decreased production of TNFα. In addition, the rs1800625 polymorphism could significantly inhibit the promoter activities of the RAGE gene., Conclusions: The rs1800625 polymorphism is a functional variant, which might be used as a relevant risk estimate for the development of sepsis and multiple organ dysfunction syndrome in patients with major trauma.

Currently DNA-based analysis of blood groups is mainly used to improve transfusion safety by reducing alloantibody formation in multiply transfused patients and by monitoring pregnancies at risk for hemolytic disease of the fetus and newborn. We present a case in which genotyping was performed after massive transfusion with unmatched group O, D- blood in a trauma setting. Our patient was genotyped as O1A1 and predicted to be D-, and we therefore transfused group A, D- red blood cell concentrates. This case demonstrates how the use of blood group genotyping in an acute setting can lead to a decrease in the unnecessary use of group O, D- blood products.

The care of multiple trauma patients has been improved through advances made in preclinical treatment, surgical procedures, and intensive care medicine. However, posttraumatic complications such as systemic inflammatory response syndrome, multiple organ dysfunction syndrome, and sepsis remain a major problem following multiple trauma. Components of the innate immune system and other inflammatory mediators (e.g., procalcitonin) play a pivotal role in the pathophysiology of posttraumatic complications. Studies investigating the genetic predisposition for complications after multiple trauma have provided evidence for a genetic heterogeneity in the posttraumatic immune response. The differences in response to multiple trauma associated with single-nucleotide polymorphisms may contribute to the development of new genetically tailored diagnostic and therapeutic interventions improving outcome in this patient population. In addition, detrimental adverse effects of adjuvant therapy could be avoided in other patients who, by genotype, are predicted not to benefit.

Purpose: To determine the clinical relevance of 13 reported common single-nucleotide polymorphisms (SNPs) of cytokine genes in patients with major trauma., Methods: Thirteen SNPs in nine key cytokine genes were selected for association study in 308 patients with major trauma on the basis of previous functional or association data. An allele-specific oligonucleotide array was developed and used to genotype 308 patients with major trauma. The clinical relevance of the 13 SNPs was assessed by observation of cytokine production and outcome of trauma patients., Results: Results from the allele-specific oligonucleotide array indicated that 8 [interleukin-1beta (IL-1beta)/-1470, IL-1beta/-511, IL-1beta/-31, IL-4/-589, IL-6/-572, IL-8/-251, IL-10/-819, and tumor necrosis factor alpha (TNFalpha)/-308] out of the 13 SNPs were associated with respective ex vivo cytokine production by peripheral leukocytes in response to lipopolysaccharide (LPS) stimulation at admission, or risk of development of sepsis or organ dysfunction in major trauma patients. Patients with more than four risk alleles of the eight SNPs had more than 50% sepsis morbidity and more severe organ dysfunction., Conclusions: Polymorphisms of IL-1beta/-1470, IL-1beta/-511, IL-1beta/-31, IL-4/-589, IL-6/-572, IL-8/-251, IL-10/-819, and TNFalpha/-308 are susceptibility loci for the development of sepsis and organ dysfunction in major trauma patients.

Recent reports have indicated that IL-1[beta] is excessively released into the circulation during sepsis, and the expression level is closely correlated with the clinical course. Polymorphisms in the promoter region of IL-1B have been shown to affect LPS-induced IL-1[beta] transcription in vitro and IL-1[beta] plasma levels in healthy adults and to confer susceptibility to inflammatory diseases. However, it is not clear whether they confer susceptibility to sepsis after major trauma. The aim of this study was to search for association of polymorphisms (-1470G/C, -511T/C, and -31C/T) in the IL-1B gene promoter with the susceptibility to sepsis in a cohort of 238 major trauma patients. Genotyping of each patient for the three single-nucleotide polymorphisms was performed by restriction fragment length polymorphism-polymerase chain reaction method. Multivariate logistic regression analysis showed that the -1470 and -31 polymorphisms were associated with IL-1[beta] production by peripheral leukocytes in response to ex vivo LPS stimulation in an allele dose-dependent manner. Moreover, trauma patients carrying the -1470G or -31T alleles were more likely to develop sepsis compared with those with the -1470C or -31C allele (P = 0.014 and P = 0.038, respectively). Of the eight possible haplotypes composed of the three loci, the GCT and CTC haplotypes were associated with significantly higher and lower IL-1[beta] secretion (P = 0.005 and P = 0.035, respectively). Moreover, the GCT haplotype imparted higher risk of sepsis after severe injury (P = 0.04; odds ratio, 1.131; 95% confidence interval, 1.013-1.678). GCT homozygote patients also showed higher multiple organ dysfunction scores than CTC homozygote patients (P = 0.048). These data suggest that the IL-1[beta] promoter polymorphisms -1470G/C, -511T/C, and -31C/T may be functional both in vitro and in vivo. It may be possible to use these polymorphisms as relevant risk estimates for sepsis in trauma patients.

Introduction: As outcome to severe trauma is frequently affected by massive blood loss and consecutive hemorrhagic shock, replacement of red blood cell (RBC) units remains indispensable. Administration of RBC units is an independent risk factor for adverse outcome in patients with trauma. The impact of massive blood transfusion or uncrossmatched blood transfusion on the patients' immune response in the early posttraumatic period remains unclear., Material: Thirteen patients presenting with blunt multiple injuries (Injury Severity Score >16) were studied. Monocytes were obtained on admission and at 6, 12, 24, 48, and 72 hours after trauma. Biotinylated complementary RNA targets were hybridized to Affymetrix HG U 133A microarrays. The data were analyzed by a supervised analysis based on whether the patients received massive blood transfusions, and then subsequently, by hierarchical clustering, and by Ingenuity pathway analysis., Results: Supervised analysis identified 224 probe sets to be differentially expressed (p < 0.001) in patients who received massive blood transfusion, when compared with those who did not. In addition, 331 probe sets were found differentially expressed (p < 0.001) in patients who received uncrossmatched RBC units in comparison with those who exclusively gained crossmatched ones. Functional pathway analysis of the respectively identified gene expression profiles suggests a contributory role by the AKT/PI3Kinase pathway, the mitogen-activated protein-kinase pathway, the Ubiquitin pathway, and the diverse inflammatory networks., Conclusion: We exhibited for the first time a serial, sequential screening analysis of monocyte messenger RNA expression patterns in patients with multiple trauma indicating a strongly significant association between the patients' genomic response in blood monocytes and massive or uncrossmatched RBC substitution.

Although MIP-1alpha is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1alpha plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1alpha-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 +/- 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum alpha-glutathione transferase, TNF-alpha, IL-6, IL-10, MCP-1, and MIP-1alpha and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1alpha KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1alpha plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1alpha, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1alpha levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.

Hypophosphatasia is a rare inherited disorder characterized by defective skeletal mineralization and low alkaline phosphatase activities in the serum. The genetic cause of hypophosphatasia is believed related to inactivating mutations in the TNSALP gene, encoding tissue-nonspecific alkaline phosphatase. Another rare inheritable disease, Saethre-Chotzen syndrome, leads to premature fusion of the cranial sutures caused by heterozygous mutations of the human TWIST1 gene. Because the two disorders apparently are not genetically related (only reported individually) yet both involve defective skeletal formation, we believe it is important to report our findings on a patient harboring mutations of TNSALP and TWIST1.

Background: Posttraumatic immune system activation in major trauma patients is linked to systemic inflammatory response syndrome, multiple organ failure (MOF), and mortality. Recent studies suggest that genome-wide expression is altered in response to distinct clinical parameters; however, the functional allocation of theses genes remains unclear., Patients and Methods: Thirteen patients after major trauma (Injury Severity Score < 16) were studied. Monocytes were obtained on admission (within 90 min) and at 6, 12, 24, 48, and 72 h after trauma. Complementary ribonucleic acid (RNA) targets were hybridized to Affymetrix HG U 133A microarrays. Searching for genes that are differentially expressed, the patients were dichotomously assigned depending upon survival, injury severity, and MOF. The data were analyzed by supervised analysis, clustering, and comparative pathway analysis., Results: Gene expression profiles of patients with adverse outcomes (763 probe sets) mainly consist of those involved in "immunological activation" or "cellular movement," whereas the gene set associated with MOF (660) is associated with "cancer" and "cell death." Injury severity (295) leads to an overexpression of genes involved in inflammatory disease., Conclusion: We demonstrate for the first time a serial, sequential screening analysis of monocyte messenger RNA expression patterns after multiple injury indicating a strongly significant connection between the patients' expression profile and different clinical parameters. The latter provoke a characteristic overexpression of specific functional gene ontologies. Further studies to clarify clinical consequence of this differential gene regulation are currently anticipated.

Introduction: Interleukin-8 (IL-8) is regarded as one of the most important mediators in the pathogenesis of Adult Respiratory Distress Syndrome (ARDS). However, knowledge regarding the influence of genetic variations within the IL-8 gene either on the development of ARDS or on IL-8 production in the traumatic setting is sparse., Patients and Methods: In this prospective cohort study, patients were included if the following criteria were fulfilled: Injury Severity Score (ISS) >16, age 18-60 years and a survival >48 h after injury. Systemic IL-8 concentrations and the polymorphisms (IL-8-251A/T) were determined. Patients were separated according to the development of ARDS (group +ARDS vs. group -ARDS) and the genotypes of the IL-8-251A/T polymorphism (genotypes A/A, A/T and T/T)., Results: Group +ARDS demonstrated significantly higher IL-8 plasma concentrations from day 3 until the end of the observation period compared to group -ARDS. In addition, duration of mechanical ventilation and length of stay in the ICU were significantly longer in this group. Furthermore, a significant association between the IL-8-251A allele and IL-8 production (day 4-8) was observed. Genotype A/A showed a significantly longer duration of mechanical ventilation compared to genotype T/T. A trend towards an association between the IL-8-251A allele and an increased incidence of posttraumatic ARDS was observed (p=0.08)., Conclusion: This data reaffirms a central role of IL-8 in the pathogenesis of ARDS. Furthermore, it points towards a genetic predisposition for posttraumatic IL-8 synthesis which might also be associated with the development of posttraumatic ARDS.

Severe trauma induces sustained changes of the immune response, which are thought to be related to secondary organ dysfunction. Despite a similar injury severity, the extent of the inflammatory response may vary between polytraumatized patients. It is unclear whether inflammatory variability is associated with genetic variations. In this prospective cohort study, patients were included when the following criteria were fulfilled: Injury Severity Score >16, age 18 to 60 years, and a survival >48 h after injury. Four different polymorphisms (TNF-Nco1, IL-1-Taq1, IL-6-174G/C, and IL-8-251A/T) were determined. Patients were separated according to the severity of the systemic inflammatory response syndrome (SIRS; ACCP/SCCM criteria: >2 criteria at 2 consecutive days or at 3 days of the observation period: group +SIRS;

Post-traumatic inflammation is connected to monocyte dysfunction characterized by reduced NF-kappaB translocation during the first post-traumatic days. Because the exact dynamic of monocytic NF-kappaB translocation in patients directly after trauma remains unclear, the aim of this pilot study was to measure the intranuclear presence of NF-kappaB in monocytes from patients with multiple injuries initially after the trauma and during the early post-traumatic period and to compare these results with downstream-placed mRNA expression alteration of TNF-alpha, as well as with clinical data. Eleven patients were enrolled with an Injury Severity Score of 16 to 66 points, and blood samples were drawn on admission within 90 min and at 6, 12, 24, 48, and 72 h after trauma. NF-kappaB translocation of monocytic nuclear protein was analyzed by electrophoretic mobility shift assay and was quantified by densitometry as arbitrary units. In addition, monocytes of healthy volunteers were analyzed either native (-, control) or after LPS stimulation (+, control). For determination of downstream mRNA encoding for TNF-alpha, quantitative reverse transcriptase-PCR was performed. For both parameters, the negative control values were set as baseline (=1) and results from positive controls and patients were given as a relative alteration ratio without unit. Initial post-traumatic NF-kappaB translocation was significantly increased in trauma patients on admission (88 +/- 37) and 6 h after trauma (59 +/- 28) compared with the baseline level. In contrast, TNF-alpha mRNA was not increased on admission (1.7 +/- 0.9) and decreased even below baseline after 12 h. The substantial information of our study arises from the analysis of the dynamic of NF-kappaB translocation of monocytes. Enabled by closely matched sequential blood sampling strictly standardized to the traumatic event, an essential increase of monocytic signal transduction and transcription could be elucidated in the very early post-traumatic period, which precedes the down-regulation of the innate immune system.

Objective: To examine the influence of genetic variations in heat shock proteins on trauma outcome., Design: Prospective, noninterventional, single-center study., Setting: Level I trauma center., Subjects: Eighty consecutive severe multiple trauma patients., Interventions: None., Measurements and Main Results: Plasma concentrations of interleukin-6 and tumor necrosis factor-alpha were measured over a 5-day course by chemiluminescence-immunoassay. The genotypes of the polymorphisms HSPA1B (HSP70-2) G1538A and HSPA1L (HSP70-Hom) C2437T were determined by polymerase chain reaction and restriction cleavage with PstlI or NcoI, respectively. Allele frequency of the HSPA1B 1538 G allele was 0.569, and that of the HSPA1L 2437 T allele was 0.821. Interleukin-6 concentrations rapidly increased and dropped to almost normal after 5 days, whereas tumor necrosis factor-alpha concentrations increased until day 5. Patients carrying the genotypes HSPA1B AG or HSPA1L CT had significantly higher plasma concentrations of tumor necrosis factor-alpha and interleukin-6 compared with those with genotype GG or TT. Presence of the HSPA1L genotype CT also was a significant risk factor to develop liver failure (odds ratio, 4.6; 95% confidence interval, 1.5-14.1) and to acquire at least one complication severe enough to score three points according to the Denver multiple organ failure score (odds ratio, 3.0; 95% confidence interval, 1.1-9.2)., Conclusion: The data indicate that genetic variations of the heat shock proteins HSPA1B and HSPA1L may contribute to clinical outcome after severe injury.

Among identified adhesion molecules, the L-selectin on neutrophils enables the first step of leukocyte adherence to activated endothelial cells. To allow firm adhesion of neutrophils, L-selectin is then split off the cell membrane. It was hypothetized that an increase of the constitutively high serum level of soluble L-selectin may indicate an ongoing pathological neutrophil sequestration to the endothelial cells associated with activation and injury of the cells. To evaluate this hypothesis, sL-selectin serum levels and neutrophil L-selectin expression of healthy volunteers (group A, n = 15), as well as of surgical patients, were investigated. Group B (n = 26) included patients subjected to elective limb surgery (mean operation time, 122 min), and group C (n = 45) comprised trauma patients. sL-selectin serum levels were measured daily over a 14-day period. Neutrophil L-selectin expression was evaluated by FACS analysis using the humanized anti-L-selectin antibody HuDreg 55 over a period of 3 days at minimum in both experimental groups. The binding of sL-selectin to endothelial cells was also examined in vitro. Elective limb surgery resulted in lower pre- and post-operative sL-selectin plasma levels (800-1,000 ng/mL) compared to healthy volunteers (1,100-1,200 ng/mL) with insignificant changes throughout the study period. Trauma patients revealed even lower sL-selectin levels (400-600 ng/mL). When these patients were discriminated by the multiple organ dysfunction (MOD) score of Moore in +MOD (n = 9, ISS = 31.7) and -MOD (n = 36, ISS = 25.0), a significant difference became evident. In +MOD patients sL-selectin levels remained on a low basis of 350 ng/mL, whereas in -MOD patients the initial low sL-selectin level subsequently rose to 800 ng/mL, similar to that of elective surgery patients. FACS analysis revealed a significant drop in neutrophil L-selectin expression 24 h after trauma compared to normal. Also, +MOD and -MOD patients were significantly discriminated by the L-selectin expression at this time. The in vitro studies revealed evidence for binding of sL-selectin to endothelial cells independently on the presence of neutrophils. According to our data, increasing severity of the post-operative/posttraumatic course is associated with decreasing sL-selectin serum levels and also reduced neutrophil L-selectin expression. In view of the in vitro results, this probably indicates competitive enhanced binding of sL-selectin to endothelial cells, thus masking the elevated activation of neutrophils and their ability for endothelial adherence.

Background: In response to traumatic injury or infection, human neutrophils are directed to the site of injury or infection by CXC chemokines that signal via 2 receptors, CXCR-1 and CXCR-2. In vitro studies have shown preferential loss of CXCR-2 expression and function after exposure to interleukin 8, N-formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and tumor necrosis factor alpha., Hypothesis: CXCR-2 expression and function are preferentially down-regulated in severely injured patients., Methods: We studied 20 patients within 24 hours of admission to the hospital. Patients with head injuries were excluded. Injury Severity Scores (range, 1-50; mean, 35) were calculated for each patient. To determine expression of CXCR-1 and CXCR-2, flow cytometry was used. Intracellular calcium mobilization and neutrophil migration to 10 nmol of interleukin 8, growth-related oncogene alpha, and fMLP was measured to determine receptor function., Results: Compared with CXCR-1, there is a greater loss of CXCR-2 receptor expression in the severely injured group (P = .01). Neutrophils from patients with Injury Severity Scores greater than 16 did not mobilize calcium in response to growth-related oncogene alpha. However, there was no loss of calcium mobilization to interleukin 8 or fMLP. Chemotaxis to various stimulants is decreased in all injury groups., Conclusions: CXCR-2 expression and function are preferentially down-regulated in severely injured patients. Our data suggest that there are multiple mechanisms, in addition to receptor down-regulation, that play a role in the loss of migration and calcium flux in human neutrophils after injury.

The study was performed to further elucidate the mechanisms of dysfunctional T-cell activation following extensive burn and mechanical injuries. The major regulatory level of interleukin-2 (IL-2) release under stressful conditions was determined via parallel analysis of IL-2 messenger RNA (mRNA) expression and IL-2 protein synthesis in mitogen-stimulated peripheral blood mononuclear cell (PBMCs) cultures on consecutive days postinjury. Furthermore, we wanted to scrutinize if inadequate lymphokine production after trauma is possibly a result of defective transduction of extracellular signals to the T-cell nucleus. Fourteen patients (11 men, 3 women, average age 38 +/- 6 years, Injury Severity Score 34 +/- 2) were included in the study. The PBMCs were isolated on days 1, 3, 5, 7, and 10 and stimulated either with the mitogen phytohemagglutinin (PHA) alone or in combination with the protein kinase C (PKC) activator phorbol myristate acetate (PMA). The protein release was examined via bioassay (human con-A blasts) from the supernatants, and the cellular RNA was indicated by radioactive hybridization with the specific complementary DNA (cDNA) after Northern blotting. The IL-2 protein synthesis generated in PHA-stimulated PBMC cultures following trauma, compared with that in controls (0.62 +/- 0.04 U/mL) was persistently and significantly depressed during the observation time with a 57% inhibition on day 10-identical with that on day 1. The Northern blot analysis of IL-2 mRNA expression in the lysates of PHA-stimulated cell cultures after trauma could not detect any mRNA signal for IL-2, in contrast to the control cells.(ABSTRACT TRUNCATED AT 250 WORDS)