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Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known to participate in immune responses and play significant functions during infections, the role of MKs within the immune system remains largely unexplored. Histological studies of sepsis patients identified increased nucleated CD61+ cells (MKs) in the lungs, and CD61+ staining (likely platelets within microthrombi) in the kidneys, which correlated with the development of organ dysfunction. Detailed imaging cytometry of peripheral blood from patients with sepsis found significantly higher MK counts, which we predict would likely be misclassified by automated hematology analyzers as leukocytes. Utilizing in vitro techniques, we show that both stem cell derived MKs (SC MKs) and cells from the human megakaryoblastic leukemia cell line, Meg-01, undergo chemotaxis, interact with bacteria, and are capable of releasing chromatin webs in response to various pathogenic stimuli. Together, our observations suggest that MK cells display some basic innate immune cell behaviors and may actively respond and play functional roles in the pathophysiology of sepsis.

Summary: Severe trauma predisposes patients to multiple independent infection episodes (MIIEs), leading to augmented morbidity and mortality. We developed a method to identify increased MIIE risk before clinical signs appear, which is fundamentally different from existing approaches entailing infections' detection after their establishment. Applying machine learning algorithms to genome-wide transcriptome data from 128 adult blunt trauma patients' (42 MIIE cases and 85 non-cases) leukocytes collected ≤48 hr of injury and ≥3 days before any infection, we constructed a 15-transcript and a 26-transcript multi-biomarker panel model with the least absolute shrinkage and selection operator (LASSO) and Elastic Net, respectively, which accurately predicted MIIE (Area Under Receiver Operating Characteristics Curve [AUROC] [95% confidence intervals, CI]: 0.90 [0.84–0.96] and 0.92 [0.86–0.96]) and significantly outperformed clinical models. Gene Ontology and network analyses found various pathways to be relevant. External validation found our model to be generalizable. Our unique precision medicine approach can be applied to a wide range of patient populations and outcomes.

Abstract Metabolic derangements are a clinically significant complication of major trauma (e.g., burn injury) and include various aspects of metabolism, such as insulin resistance, muscle wasting, mitochondrial dysfunction and hyperlactatemia. Nonetheless, the molecular pathogenesis and the relation between these diverse metabolic alterations are poorly understood. We have previously shown that burn increases farnesyltransferase (FTase) expression and protein farnesylation and that FTase inhibitor (FTI) prevents burn-induced hyperlactatemia, insulin resistance, and increased proteolysis in mouse skeletal muscle. In this study, we found that burn injury activated mTORC1 and hypoxia-inducible factor (HIF)-1α, which paralleled dysfunction, morphological alterations (i.e., enlargement, partial loss of cristae structure) and impairment of respiratory supercomplex assembly of the mitochondria, and ER stress. FTI reversed or ameliorated all of these alterations in burned mice. These findings indicate that these burn-induced changes, which encompass various aspects of metabolism, may be linked to one another and require protein farnesylation. Our results provide evidence of involvement of the mTORC1-HIF-1α pathway in burn-induced metabolic derangements. Our study identifies protein farnesylation as a potential hub of the signaling network affecting multiple aspects of metabolic alterations after burn injury and as a novel potential molecular target to improve the clinical outcome of severely burned patients.

Introduction: The advent of digital slides offers new opportunities within the practice of pathology such as the use of image analysis techniques to facilitate computer aided diagnosis (CAD) solutions. Use of CAD holds promise to enable new levels of decision support and allow for additional layers of quality assurance and consistency in rendered diagnoses. However, the development and testing of prostate cancer CAD solutions requires a ground truth map of the cancer to enable the generation of receiver operator characteristic (ROC) curves. This requires a pathologist to annotate, or paint, each of the malignant glands in prostate cancer with an image editor software - a time consuming and exhaustive process.

Sepsis remains the major cause of death in patients with major burn injuries. In the present investigation we evaluated the interaction between burn injuries of varying severity and preexisting distant infection. We used Gram-negative bacteria (Pseudomonas aeruginosa and Proteus mirabilis) that were genetically engineered to be bioluminescent, which allowed for noninvasive, sequential optical imaging of the extent and severity of the infection. The bioluminescent bacteria migrated from subcutaneous abscesses in the leg to distant burn wounds on the back depending on the severity of the burn injury, and this migration led to increased mortality of the mice. Treatment with ciprofloxacin, injected either in the leg with the bacterial infection or into the burn eschar, prevented this colonization of the wound and decreased mortality. The present data suggest that burn wounds can readily become colonized by infections distant from the wound itself.

Mesenchymal stem cell (MSC) transplantation has been explored as a new clinical approach to repair injured tissue. A growing corpus of studies have highlighted two important aspects of MSC therapy: 1) MSCs can modulate T-cell-mediated immunological responses, and ( 2 ) systemically administered MSCs home to sites of ischemia or injury. In this review, we describe the known mechanisms of immunomodulation and homing of MSCs. First, we examine the low immunogenicity of MSCs and their antigen presentation capabilities. Next, we discuss the paracrine interactions between MSCs and innate [dendritic cells (DC)] and adaptive immune cells (T lymphocytes) with a focus on prostaglandin E 2 (PGE 2 ), indoleamine 2,3-dioxygenase (IDO), and toll-like receptor (TLR) signaling pathways. We transition to outline the steps of activation, rolling/adhesion, and transmigration of MSCs into target tissues during inflammatory or ischemic conditions. These aspects of MSC grafts—immunomodulation and homing—are contextualized to understand a reported side effect of MSC therapy, cancer development.

Bone marrow mesenchymal stromal cells (MSCs) suppress immune cell responses and have beneficial effects in various inflammatory-related immune disorders. A therapeutic modality for systemic inflammation and its consequences is not available yet. Thus, this work investigates the therapeutic effects of MSCs in injury models induced by lipopolysaccharide (LPS) or burn. Gene expression was analyzed in MSCs when exposed to inflammatory serum from injured animals and it showed remarkable alterations compared to normal culture. In addition, injured animals were transplanted intramuscularly with MSCs. Forty-eight hours after cell transplantation, kidney, lung, and liver were analyzed for infiltration of inflammatory cells and TUNEL-expressing cells. Results showed that MSCs attenuate injury by reducing the infiltration of inflammatory cells in various target organs and by reducing cell death. These data suggest that MSCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for systemic inflammatory-based disorders.

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (−1°C/min) up to −80°C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.

Quantification of the dependence of cellular oxygen uptake rate (OUR) on oxygen partial pressure is useful for the design and testing of bioartificial devices which utilize cells. Thus far, this information has only been obtained from suspended cells and from cells attached to microcarriers. In this work, a device was developed to obtain the dependence of OUR on oxygen partial pressure for anchorage-dependent cells cultured in standard culture dishes. The device is placed and sealed on the top of the culture dish, and holds a Clark polarographic mini-electrode flush with the bottom surface of the device. It also houses a motor to spin a magnetic stir bar within the cell chamber to insure that the medium is well-mixed. Several characteristics of the device — such as oxygen leakage into the device chamber, electrode-lag time, and linearity of the electrode at low oxygen partial pressures-were quantified and their potential effect on the values of V m (maximal OUR) and K 0.5 (oxygen partial pressure at which OUR is half-maximal) were evaluated. Comparison of V m and K 0.5 values obtained with this device with previously published values for suspended rat hepatocytes, Bacillus cereus, and E. coli indicated that the technique provides values accurate within 30% as long as the cell under study has a K 0.5 greater than approximately 1.0 mmHg. For hepatocytes cultured on 0.05 mm thickness collagen gel for 1 day (n = 4) and 3 days (n = 6), V m was found to be 0.38 ± 0.12 and 0.25 ± 0.09 nmol O 2 /s/106 cells, respectively, and K 0.5 was found to be 5.6 ± 0.5 and 3.3 ± 0.6 mmHg, respectively. This technique should aid in predicting bioreactor conditions such as flow rate, cell density, distance of cell from flow, and gas phase oxygen partial pressure which can lead to oxygen limitations. In addition, further studies of the effect of factors such as extracellular matrix composition, metabolic substrate, and drugs on the dependence of OUR on oxygen partial pressure for many anchorage-dependent cell types can be pursued with this technique.

Despite more than 30 yr of research and development, an artificial liver has still not yet become clinical reality. Although previous attempts using a multiplicity of techniques including hemodialysis, hemoperfusion, plasma exchange, extracorporeal perfusion, and crosshemodialysis have shown minor improvement in patients with acute hepatic failure, limited clinical trials have failed to demonstrate any survival benefit. Encouraged by the progress on techniques that maintain long-term cultures of hepatocytes, more recent efforts have been directed at the use of hepatocytes as the basis of liver support. This review takes a critical look at past and present concepts in the development of artificial liver supports and both qualitatively and quantitatively evaluates the advantages and disadvantages of the available methodology.

Hepatocyte cryopreservation is essential to ensure a ready supply of cells for use in transplantation or as part of an extracorporeal liver assist device to provide on-demand liver support. To date, most of the work on hepatocyte cryopreservation has been performed on isolated hepatocytes, and has generally yielded cells which display low viability and greatly reduced short-term function. This report presents the development of a freezing procedure for hepatocytes cultured in a sandwich configuration. A specially designed freezing unit was used to provide controlled temperatures throughout the freeze-thaw cycle. Cooling rate, warming rate, and final freezing temperature were evaluated as to their effect on hepatocyte function as judged by albumin secretion. Under optimized conditions (cooling at 5°C/min and warming at ≥400°C/min), freezing to −40°C resulted in full recovery of albumin secretion within 2-3 days post-freezing, whereafter albumin secretion levels remained normal for the duration of the experiments (2 wks). Freezing to −80°C lead to an approximate 70% recovery of long-term protein secretion when compared to control cultures. In addition, the overall hepatocyte morphology as judged by light microscopy, closely followed the functional results. The sandwich culture configuration, thus, enables hepatocytes to maintain a satisfactory level of long-term protein secretion after a freeze-thaw cycle under optimized conditions, and offers an attractive tool for further studies into the mechanisms of freezing injury and subsequent hepatocellular recovery. These results are a promising step in the development of satisfactory storage procedures for hepatocytes.

Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4+ T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4+ T lymphocytes with high efficiency directly from 10 microlitres of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R2 = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings. [ABSTRACT FROM AUTHOR]

Trauma patients are at risk of repeated hospital-acquired infections, however predictive scores aiming to identify susceptibility to such infections are lacking. The objective of this study was to investigate whether commonly employed disease-severity scores can successfully predict susceptibility to multiple independent infectious episodes (MIIEs) among trauma patients. A secondary analysis of data derived from the prospective, longitudinal study "Inflammation and the Host Response to Injury" ("Glue Grant") was performed. 1,665 trauma patients, older than 16, were included. Patients who died within seven days from the time of injury were excluded. Five commonly used disease-severity scores [Denver, Marshall, Acute Physiology and Chronic Health Evaluation II (APACHE II), Injury Severity Score (ISS), and New Injury Severity Score (NISS)] were examined as independent predictors of susceptibility to MIIEs. The latter was defined as two or more independent infectious episodes during the index hospital stay. Multivariable logistic regression was used for the statistical analysis. 22.58% of the population was found to be susceptible to MIIEs. Denver and Marshall scores were highly predictive of the MIIE status. For every 1-unit increase in the Denver or the Marshall score, there was a respective 15% (Odds Ratio:1.15; 95% CI: 1.07-1.24; p < 0.001) or 16% (Odds Ratio:1.16; 95% CI: 1.09-1.24; p < 0.001) increase in the odds of MIIE occurrence. APACHE II, ISS, and NISS were not independent predictors of susceptibility to MIIEs. In conclusion, the Denver and Marshall scores can reliably predict which trauma patients are prone to MIIEs, prior to any clinical sign of infection. Early identification of these individuals would potentially allow the implementation of rapid, personalized, preventative measures, thus improving patient outcomes and reducing healthcare costs.

PurposeMortality and morbidity rates of elderly burn patients remain high despite numerous advancements in modern burn care. While prior studies have offered first insights on the biochemical changes in elderly burn patients compared to adults, the underlying cellular responses remain largely unknown. In this study, we aim to characterize the transcriptome of elderly burn patients and compare it to adult burn patients to obtain insights into the underlying molecular responses post-burn and to elucidate the effect of advanced age on the acute burn response.Materials and methodsMicroarray data obtained from the Glue Grant Trauma-Related Database was obtained from blood specimens for ten elderly patients (n = 10), each with a set of two sex and total body surface area (TBSA) matched adult controls (n = 20), during the acute phase post-burn. Adult and elderly demographics and clinical outcomes were contrasted using using the Chi-Square test, Fisher's Exact Test, or two-sample t-tests, as appropriate (pResultsSupervised analysis identified multiple genes that were differentially expressed between the elderly and adult groups. Pathway analysis and heatmap generation suggest that elderly patients share a distinct hypo-inflammatory response in the acute post-burn phase with downregulation of a number of immune-related pathways, including those related to antigen processing, specifically via MHC class I, ubiquitination and proteasome degradation (pConclusionsThe altered inflammatory and immune responses at the transcriptome level in elderly patients after burn are indicative of a failure in elderly burn patients to initiate an appropriate inflammatory and stress response during the acute phase post-burn.

Inflammation and apoptosis develop in skeletal muscle after major trauma, including burn injury, and play a pivotal role in insulin resistance and muscle wasting. We and others have shown that inducible nitric oxide synthase (iNOS), a major mediator of inflammation, plays an important role in stress (e.g., burn)-induced insulin resistance. However, it remains to be determined how iNOS induces insulin resistance. Moreover, the interrelation between inflammatory response and apoptosis is poorly understood, although they often develop simultaneously. Nuclear factor (NF)-κB and p53 are key regulators of inflammation and apoptosis, respectively. Sirt1 inhibits p65 NF-κB and p53 by deacetylating these transcription factors. Recently, we have shown that iNOS induces S-nitrosylation of Sirt1, which inactivates Sirt1 and thereby increases acetylation and activity of p65 NF-κB and p53 in various cell types, including skeletal muscle cells. Here, we show that iNOS enhances burn-induced inflammatory response and apoptotic change in mouse skeletal muscle along with S-nitrosylation of Sirt1. Burn injury induced robust expression of iNOS in skeletal muscle and gene disruption of iNOS significantly inhibited burn-induced increases in inflammatory gene expression and apoptotic change. In parallel, burn increased Sirt1 S-nitrosylation and acetylation and DNA-binding capacity of p65 NF-κB and p53, all of which were reversed or ameliorated by iNOS deficiency. These results indicate that iNOS functions not only as a downstream effector but also as an upstream enhancer of burn-induced inflammatory response, at least in part, by Sirt1 S-nitrosylation-dependent activation (acetylation) of p65 NF-κB. Our data suggest that Sirt1 S-nitrosylation may play a role in iNOS-mediated enhanced inflammatory response and apoptotic change, which, in turn, contribute to muscle wasting and supposedly to insulin resistance after burn injury.

Previous studies from our laboratories revealed a reduced rate of whole-blood (WB) glutathione (GSH) synthesis in severely burned patients. To determine whether WB GSH metabolism is an indicator of the status of GSH metabolism in one or more of the major organs, we used a burn rabbit model to determine GSH concentrations and rates of synthesis in WB, liver, lungs, kidney, and skeletal muscle. L-[1- 13 C]-cysteine was infused intravenously for 6 h in rabbits at 3 days post-burn and in sham burn controls. WB and organ 13 C-enrichment of cysteine and GSH was determined by gas chromatography/mass spectrometry. Plasma cysteine metabolic flux was increased significantly (P < 0.01) following burn injury. WB, liver, and lung GSH concentrations (P = 0.054, P < 0.05, and P < 0.05, respectively) and fractional rates of GSH synthesis (P < 0.05, P< 0.01, and P< 0.05, respectively) were reduced at 3 days post-burn. Kidney was unaffected. There also appears to be an increased rate of GSH transport out of the liver after burn injury. Hence, there is a differential impact of burn injury on tissue and organ GSH status, with WB qualitatively reflecting the changes in lung and liver. It will be important to determine whether these changes are due to alterations in the intrinsic capacity for GSH synthesis and/or availability of amino acid precursors of GSH.

Severe burn injury renders patients susceptible to multiple infection episodes, however identifying specific patient groups at high risk remains challenging. Burn-induced inflammatory response dramatically modifies the levels of various cytokines. Whether these changes could predict susceptibility to infections remains unknown. The aim of this study was to determine the early changes in the pro- to anti-inflammatory cytokine ratio and investigate its ability to predict susceptibility to repeated infections after severe burn trauma. The patient population consisted of 34 adult patients having early (≤48 hours since injury) blood draws following severe (≥20% total burn surface area (TBSA)) burn injury, and suffering from a first infection episode at least one day after blood collection. Plasma TNF-α and IL-10 levels were measured to explore the association between the TNF-α/IL-10 ratio, hypersusceptibility to infections, burn size (TBSA), and common severity scores (Acute Physiology and Chronic Health Evaluation (APACHEII), Baux, modified Baux (R-Baux), Ryan Score, Abbreviated Burn Severity Index (ABSI)). TNF-α/IL10 plasma ratio measured shortly after burn trauma was inversely correlated with burn size and the injury severity scores investigated, and was predictive of repeated infections (≥3 infection episodes) outcome (AUROC [95%CI] of 0.80 [0.63–0.93]). Early measures of circulating TNF-α/IL10 ratio may be a previously unidentified biomarker associated with burn injury severity and predictive of the risk of hypersusceptibility to repeated infections.

Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens.

OBJECTIVE:Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle. METHODS:A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated. RESULTS:Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277. CONCLUSIONS:Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients.

Finely tuned to respond quickly to infections, neutrophils have amazing abilities to migrate fast and efficiently towards sites of infection and inflammation. Although neutrophils ability to migrate is perturbed in patients after major burns, no correlations have yet been demonstrated between altered migration and higher rate of infections and sepsis in these patients when compared to healthy individuals. To probe if such correlations exist, we designed microfluidic devices to quantify the neutrophil migration phenotype with high precision. Inside these devices, moving neutrophils are confined in channels smaller than the neutrophils and forced to make directional decisions at bifurcations and around posts. We employed these devices to quantify neutrophil migration across 18 independent parameters in 74 blood samples from 13 patients with major burns and 3 healthy subjects. Blinded, retrospective analysis of clinical data and neutrophil migration parameters revealed that neutrophils isolated from blood samples collected during sepsis migrate spontaneously inside the microfluidic channels. The spontaneous neutrophil migration is a unique phenotype, typical for patients with major burns during sepsis and often observed one or two days before the diagnosis of sepsis is confirmed. The spontaneous neutrophil migration phenotype is rare in patients with major burns in the absence of sepsis, and is not encountered in healthy individuals. Our findings warrant further studies of neutrophils and their utility for early diagnosing and monitoring sepsis in patients after major burns.

Mitochondria integrate distinct signals that reflect specific threats to the host, including infection, tissue damage, and metabolic dysfunction; and play a key role in insulin resistance. We have found that the Pseudomonas aeruginosa quorum sensing infochemical, 2-amino acetophenone (2-AA), produced during acute and chronic infection in human tissues, including in the lungs of cystic fibrosis (CF) patients, acts as an interkingdom immunomodulatory signal that facilitates pathogen persistence, and host tolerance to infection. Transcriptome results have led to the hypothesis that 2-AA causes further harm to the host by triggering mitochondrial dysfunction in skeletal muscle. As normal skeletal muscle function is essential to survival, and is compromised in many chronic illnesses, including infections and CF-associated muscle wasting, we here determine the global effects of 2-AA on skeletal muscle using high-resolution magic-angle-spinning (HRMAS), proton ((1)H) nuclear magnetic resonance (NMR) metabolomics, in vivo (31)P NMR, whole-genome expression analysis and functional studies. Our results show that 2-AA when injected into mice, induced a biological signature of insulin resistance as determined by (1)H NMR analysis-, and dramatically altered insulin signaling, glucose transport, and mitochondrial function. Genes including Glut4, IRS1, PPAR-γ, PGC1 and Sirt1 were downregulated, whereas uncoupling protein UCP3 was up-regulated, in accordance with mitochondrial dysfunction. Although 2-AA did not alter high-energy phosphates or pH by in vivo (31)P NMR analysis, it significantly reduced the rate of ATP synthesis. This affect was corroborated by results demonstrating down-regulation of the expression of genes involved in energy production and muscle function, and was further validated by muscle function studies. Together, these results further demonstrate that 2-AA, acts as a mediator of interkingdom modulation, and likely effects insulin resistance associated with a molecular signature of mitochondrial dysfunction in skeletal muscle. Reduced energy production and mitochondrial dysfunctional may further favor infection, and be an important step in the establishment of chronic and persistent infections.

Trauma is the number one killer of individuals 1-44 y of age in the United States. The prognosis and treatment of inflammatory complications in critically injured patients continue to be challenging, with a history of failed clinical trials and poorly understood biology. New approaches are therefore needed to improve our ability to diagnose and treat this clinical condition.We conducted a large-scale study on 168 blunt-force trauma patients over 28 d, measuring ∼400 clinical variables and longitudinally profiling leukocyte gene expression with ∼800 microarrays. Marshall MOF (multiple organ failure) clinical score trajectories were first utilized to organize the patients into five categories of increasingly poor outcomes. We then developed an analysis framework modeling early within-patient expression changes to produce a robust characterization of the genomic response to trauma. A quarter of the genome shows early expression changes associated with longer-term post-injury complications, captured by at least five dynamic co-expression modules of functionally related genes. In particular, early down-regulation of MHC-class II genes and up-regulation of p38 MAPK signaling pathway were found to strongly associate with longer-term post-injury complications, providing discrimination among patient outcomes from expression changes during the 40-80 h window post-injury.The genomic characterization provided here substantially expands the scope by which the molecular response to trauma may be characterized and understood. These results may be instrumental in furthering our understanding of the disease process and identifying potential targets for therapeutic intervention. Additionally, the quantitative approach we have introduced is potentially applicable to future genomics studies of rapidly progressing clinical conditions.ClinicalTrials.gov NCT00257231

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72-120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.

Despite recent advances in our understanding the pathophysiology of trauma, the basis of the predisposition of trauma patients to infection remains unclear. A Drosophila melanogaster/Pseudomonas aeruginosa injury and infection model was used to identify host genetic components that contribute to the hyper-susceptibility to infection that follows severe trauma. We show that P. aeruginosa compromises skeletal muscle gene (SMG) expression at the injury site to promote infection. We demonstrate that activation of SMG structural components is under the control of cJun-N-terminal Kinase (JNK) Kinase, Hemipterous (Hep), and activation of this pathway promotes local resistance to P. aeruginosa in flies and mice. Our study links SMG expression and function to increased susceptibility to infection, and suggests that P. aeruginosa affects SMG homeostasis locally by restricting SMG expression in injured skeletal muscle tissue. Local potentiation of these host responses, and/or inhibition of their suppression by virulent P. aeruginosa cells, could lead to novel therapies that prevent or treat deleterious and potentially fatal infections in severely injured individuals.

Introduction: Historically, effective clinical utilization of image analysis and pattern recognition algorithms in pathology has been hampered by two critical limitations: 1) the availability of digital whole slide imagery data sets and 2) a relative domain knowledge deficit in terms of application of such algorithms, on the part of practicing pathologists. With the advent of the recent and rapid adoption of whole slide imaging solutions, the former limitation has been largely resolved. However, with the expectation that it is unlikely for the general cohort of contemporary pathologists to gain advanced image analysis skills in the short term, the latter problem remains, thus underscoring the need for a class of algorithm that has the concurrent properties of image domain (or organ system) independence and extreme ease of use, without the need for specialized training or expertise. Results: In this report, we present a novel, general case pattern recognition algorithm, Spatially Invariant Vector Quantization (SIVQ), that overcomes the aforementioned knowledge deficit. Fundamentally based on conventional Vector Quantization (VQ) pattern recognition approaches, SIVQ gains its superior performance and essentially zero-training workflow model from its use of ring vectors, which exhibit continuous symmetry, as opposed to square or rectangular vectors, which do not. By use of the stochastic matching properties inherent in continuous symmetry, a single ring vector can exhibit as much as a millionfold improvement in matching possibilities, as opposed to conventional VQ vectors. SIVQ was utilized to demonstrate rapid and highly precise pattern recognition capability in a broad range of gross and microscopic use-case settings. Conclusion: With the performance of SIVQ observed thus far, we find evidence that indeed there exist classes of image analysis/pattern recognition algorithms suitable for deployment in settings where pathologists alone can effectively incorporate their use into clinical workflow, as a turnkey solution. We anticipate that SIVQ, and other related class-independent pattern recognition algorithms, will become part of the overall armamentarium of digital image analysis approaches that are immediately available to practicing pathologists, without the need for the immediate availability of an image analysis expert.