An Integrated Clinico-transcriptomic Approach Identifies a Central Role of the Heme Degradation Pathway for Septic Complications after Trauma.

OBJECTIVE: The present study was aimed to identify mechanisms linked to complicated courses and adverse events after severe trauma by a systems biology approach. SUMMARY BACKGROUND DATA: In severe trauma, overwhelming systemic inflammation can result in additional damage and the development of complications, including sepsis. METHODS: In a prospective, longitudinal single-center study, RNA samples from circulating leukocytes from patients with multiple injury (injury severity score ≥17 points; n = 81) were analyzed for dynamic changes in gene expression over a period of 21 days by whole-genome screening (discovery set; n = 10 patients; 90 samples) and quantitative RT-PCR (validation set; n = 71 patients, 517 samples). Multivariate correlational analysis of transcripts and clinical parameters was used to identify mechanisms related to sepsis. RESULTS: Transcriptome profiling of the discovery set revealed the strongest changes between patients with either systemic inflammation or sepsis in gene expression of the heme degradation pathway. Using quantitative RT-PCR analyses (validation set), the key components haptoglobin (HP), cluster of differentiation (CD) 163, heme oxygenase-1 (HMOX1), and biliverdin reductase A (BLVRA) showed robust changes following trauma. Upregulation of HP was associated with the severity of systemic inflammation and the development of sepsis. Patients who received allogeneic blood transfusions had a higher incidence of nosocomial infections and sepsis, and the amount of blood transfusion as source of free heme correlated with the expression pattern of HP. CONCLUSIONS: These findings indicate that the heme degradation pathway is associated with increased susceptibility to septic complications after trauma, which is indicated by HP expression in particular.

The glycolytic enzyme enolase represents a plasminogen-binding protein on the surface of a wide variety of medically important fungal species.

Allergies are an increasing issue in human health and can, eventually, cause severe anaphylactic shock. Aspergillus fumigatus and Candida albicans are leading causes of life-threatening invasive fungal infections in immunocompromised patients, but can also cause severe allergic responses in otherwise healthy individuals. The glycolytic enzyme enolase is known as a major allergen despite its function in intracellular metabolism. Therefore, its presentation on surfaces of different fungal species was investigated by using antibodies raised against recombinant enolases from A. fumigatus and C. albicans. Examination of antibody specificity revealed cross-reactivity to cell-free extracts from Aspergillus terreus, Aspergillus flavus, Aspergillus nidulans and Candida glabrata, but not against any of the three human enolases. Antibody specificity was further confirmed by hybridization with other recombinant fungal enolases, where the antibodies recognized different subsets of fungal enolases. When surface presentation of enolase was tested on intact fungal cells, a positive staining was obtained with those antibodies that also recognized the enzyme from the respective cell-free extract. This implies a general surface presentation of this glycolytic enzyme among fungal species and provides hints for its predominant recognition as an allergen. Additionally, A. fumigatus and C. albicans enolase bound to human plasminogen, which remained accessible for the plasminogen activator uPA. This implies a potential role of enolase in the invasion and dissemination process during fungal infections.

Improvement of prognostic performance in severely injured patients by integrated clinico-transcriptomics: a translational approach.

INTRODUCTION: Severe trauma triggers a systemic inflammatory response that contributes to secondary complications, such as nosocomial infections, sepsis or multi-organ failure. The present study was aimed to identify markers predicting complications and an adverse outcome of severely injured patients by an integrated clinico-transcriptomic approach. METHODS: In a prospective study, RNA samples from circulating leukocytes from severely injured patients (injury severity score ≥ 17 points; n = 104) admitted to a Level I Trauma Center were analyzed for dynamic changes in gene expression over a period of 21 days by quantitative RT-PCR. Transcriptomic candidates were selected based on whole genome screening of a representative discovery set (n = 10 patients) or known mechanisms of the immune response, including mediators of inflammation (IL-8, IL-10, TNF-α, MIF, C5, CD59, SPHK1), danger signaling (HMGB1, TLR2, CD14, IL-33, IL-1RL1), and components of the heme degradation pathway (HP, CD163, HMOX1, BLVRA, BLVRB). Clinical markers comprised standard physiological and laboratory parameters and scoring systems routinely determined in trauma patients. RESULTS: Leukocytes, thrombocytes and the expression of sphingosine kinase-1 (SPHK1), complement C5, and haptoglobin (HP) have been identified as markers with the best performance. Leukocytes showed a biphasic course with peaks on day 0 and day 11 after trauma, and patients with sepsis exhibited significantly higher leukocyte levels. Thrombocyte numbers showed a typical profile with initial thrombopenia and robust thrombocytosis in week 3 after trauma, ranging 2- to 3-fold above the upper normal value. 'Relative thrombocytopenia' was associated with multi-organ dysfunction, the development of sepsis, and mortality, the latter of which could be predicted within 3 days prior to the time point of death. SPHK1 expression at the day of admission indicated mortality with excellent performance. C5-expression on day 1 after trauma correlated with an increased risk for the development of nosocomial infections during the later course, while HP was found to be a marker for the development of sepsis. CONCLUSIONS: The combination of clinical and transcriptomic markers improves the prognostic performance and may represent a useful tool for individual risk stratification in trauma patients.

HES 130/0.4 impairs haemostasis and stimulates pro-inflammatory blood platelet function.

INTRODUCTION: Hydroxyethyl starch (HES) solutions are widely used for volume replacement therapy but are also known to compromise coagulation, impair renal function and increase long-term mortality. To test the hypotheses that HES 130/0.4 has fewer adverse effects than HES 200/0.5 and exerts anti-inflammatory properties, we compared the effects of HES 130/0.4, HES 200/0.5 and saline on in vitro haemostasis and pro-inflammatory platelet function. METHODS: Whole blood samples from healthy volunteers were mixed with 6% HES 130/0.4, 10% HES 200/0.5, or normal saline to achieve a final haemodilution rate of 10% or 40%. Haemostatic capacity was characterised by thromboelastography (ROTEM) and measurement for FXIIIa activity. Platelet activation and pro-inflammatory platelet functions were characterised by flow cytometry measuring the platelet activation marker CD62P and binding of fibrinogen to platelets as well as the formation of heterotypic platelet-leukocyte conjugates. RESULTS: Compared with saline, HES 130/0.4 dose-dependently impaired formation and firmness of the fibrin clot but did not affect the fibrin crosslinking activity of FXIIIa. At 40% but not at 10% haemodilution rate, HES 200/0.5 also increased platelet fibrinogen binding and both HES solutions increased expression of CD62P, the main receptor for platelet-leukocyte adhesion. HES 130/0.4 but not HES 200/0.5 increased formation of platelet-neutrophil conjugates and, to a lesser degree, platelet-monocyte conjugates. CONCLUSIONS: Our data demonstrate that HES 130/0.4 has similar adverse effects as HES 200/0.5. In particular, both types of HES impair coagulation capacity and stimulate, rather than attenuate, pro-inflammatory platelet function.

Molecular signatures of liver dysfunction are distinct in fungal and bacterial infections in mice.

Rationale: The liver is a central organ not only for metabolism but also immune function. Life-threatening infections of both bacterial and fungal origin can affect liver function but it is yet unknown whether molecular changes differ depending on the pathogen. We aimed to determine whether the hepatic host response to bacterial and fungal infections differs in terms of hepatic metabolism and liver function. Methods: We compared murine models of infection, including bacterial peritoneal contamination and infection (PCI), intraperitoneal and systemic C. albicans infection, at 6 and 24 h post-infection, to sham controls. The molecular hepatic host response was investigated by the detection of regulatory modules based on large-scale protein-protein interaction networks and expression data. Topological analysis of these regulatory modules was used to reveal infection-specific biological processes and molecular mechanisms. Intravital microscopy and immunofluorescence microscopy were used to further analyze specific aspects of pathophysiology such as cholestasis. Results: Down-regulation of lipid catabolism and bile acid synthesis was observed after 6 h in all infection groups. Alterations in lipid catabolism were characterized by accumulation of long chain acylcarnitines and defective beta-oxidation, which affected metabolism by 6 h. While PCI led to an accumulation of unconjugated bile acids (BA), C. albicans infection caused accumulation of conjugated BA independent of the route of infection. Hepatic dye clearance and transporter expression revealed reduced hepatic uptake in fungal infections vs. defects in secretion following polybacterial infection. Conclusion: Molecular phenotypes of lipid accumulation and cholestasis allow differentiation between pathogens as well as routes of infection at early stages in mice. Targeted metabolomics could be a useful tool for the profiling of infected/septic patients and the type of pathogen, with subsequent customization and targeting of therapy.