Florian Uhle

Methylglyoxal as a new biomarker in patients with septic shock: an observational clinical study

IntroductionThe role of reactive carbonyl species, such as methylglyoxal (MG), has been overlooked within the context of the sepsis syndrome. The aims of this study were to assess the impact of MG formation in different inflammatory settings and to evaluate its use for early diagnosis as well as prognosis of the sepsis syndrome.MethodsIn total, 120 patients in three groups were enrolled in this observational clinical pilot study. The three groups included patients with septic shock (nu2009=u200960), postoperative controls (nu2009=u200930), and healthy volunteers (nu2009=u200930). Plasma samples from patients with septic shock were collected at sepsis onset and after 24xa0hours and 4, 7, 14, and 28xa0days. Plasma samples from postoperative controls were collected prior to surgery, immediately following the end of the surgical procedure as well as 24xa0hours later and from healthy volunteers once. Plasma levels of MG were determined by high-performance liquid chromatography. Additionally, plasma levels of procalcitonin, C-reactive protein, soluble CD14 subtype, and interleukin-6 were determined.ResultsPatients with septic shock showed significantly higher plasma levels of MG at all measured times, compared with postoperative controls. MG was found to identify patients with septic shock more effectively—area under the curve (AUC): 0.993—than procalcitonin (AUC: 0.844), C-reactive protein (AUC: 0.791), soluble CD14 subtype (AUC: 0.832), and interleukin-6 (AUC: 0.898) as assessed by receiver operating characteristic (ROC) analysis. Moreover, plasma levels of MG in non-survivors were significantly higher than in survivors (sepsis onset: *Pu2009=u20090.018 for 90-day survival; **Pu2009=u20090.008 for 28-day survival). Plasma levels of MG proved to be an early predictor for survival in patients with septic shock (sepsis onset: ROC-AUC 0.710 for 28-day survival; ROC-AUC 0.686 for 90-day survival).ConclusionsMG was identified as a marker for monitoring the onset, development, and remission of sepsis and was found to be more useful than routine diagnostic markers. Further studies are required to determine the extent of MG modification in sepsis and whether targeting this pathway could be therapeutically beneficial to the patient.Trial registrationGerman Clinical Trials Register DRKS00000505. Registered 8 November 2010.

Cardiac effects of echinocandins after central venous administration in adult rats

ABSTRACT Echinocandins have become the agents of choice for early and specific antifungal treatment in critically ill patients. In vitro studies and clinical case reports revealed a possible impact of echinocandin treatment on cardiac function. The aim of our study was to evaluate echinocandin-induced cardiac failure. Using an in vivo rat model, we assessed hemodynamic parameters and time to hemodynamic failure after central venous application (vena jugularis interna) of anidulafungin (low-dose group, 2.5 mg/kg body weight [BW]; high-dose group, 25 mg/kg BW), caspofungin (low-dose group, 0.875 mg/kg BW; high-dose group, 8.75 mg/kg BW), micafungin (low-dose group, 3 mg/kg BW; high-dose group, 30 mg/kg BW), and placebo (0.9% sodium chloride). Left ventricular heart tissue was collected to determine mitochondrial enzyme activity via spectrophotometric measurements. mRNA expression of transcriptional regulators and primary mitochondrial transcripts, mitochondrial DNA (mtDNA) content, and citrate synthase activity were also explored. Animals receiving high-dose anidulafungin or caspofungin showed an immediate decrease in hemodynamic function. All of the subjects in these groups died during the observation period. Every animal in the untreated control group survived (P < 0.001). Hemodynamic failure was not noticed in the anidulafungin and caspofungin low-dose groups. Micafungin had no impact on cardiac function. In analyzing mitochondrial enzyme activity and mitochondrial transcripts, we found no association between echinocandin administration and the risk for hemodynamic failure. Further experimental studies are needed to elucidate the underlying mechanisms involved in cardiotoxic echinocandin effects. In addition, randomized controlled clinical trials are needed to explore the clinical impact of echinocandin treatment in critically ill patients.

Role of the RAGE Axis during the Immune Response after Severe Trauma: A Prospective Pilot Study.

Background. Severe traumatization induces a complex pathophysiology, driven by the patients own immune system. The initial activation is a result of damage-associated molecular patterns, which are released from disrupted and dying cells and recognized by immune receptors, for example, RAGE. In this study we aimed to evaluate the contribution of the RAGE axis to early and late immune responses. Methods. We enrolled 16 patients with severe trauma together with 10 patients after major abdominal surgery and 10 healthy volunteers. Blood samples were taken on admission and every 48 h for a total of 8 days. Plasma concentrations of various RAGE ligands as well as RAGE isoforms and IL-6 were measured by ELISA. Monocyte surface expression of RAGE and HLA-DR was assessed by flow cytometry. Results. High and transient levels of IL-6 and methylglyoxal characterize the early immune response after trauma, whereas samples from later time points provide evidence for a secondary release of RAGE ligands. Conclusion. Our results provide evidence for a persisting activation of the RAGE axis while classical mediators like IL-6 disappear early. Considering the immunocompromised phenotype of the monocytes, the RAGE ligands might be substantial contributors to the well-known secondary stage of impaired immune responsiveness in trauma patients.

GTS-21 reduces microvascular permeability during experimental endotoxemia

INTRODUCTIONnNo effective pharmacological therapy is currently available to attenuate tissue edema formation due to increased microvascular permeability in sepsis. Cholinergic mediators have been demonstrated to exert anti-inflammatory effects via the α7 nicotinic acetylcholine receptor (α7nAChR) during inflammation. GTS-21, a partial α7nAChR agonist, is an appealing therapeutic substance for sepsis-induced microvascular inflammation due to its demonstrated cholinergic anti-inflammatory properties and its favorable safety profile in clinical trials. This study evaluated the effect of GTS-21 on microvascular permeability and leukocyte adhesion during experimental endotoxemia.nnnMETHODSnMale Wistar rats (n=60) were anesthetized and prepared for intravital microscopy (IVM). Sevoflurane inhalation combined with propofol (10mg/kg) and fentanyl (5μg/kg) was used for anesthesia induction, followed by continuous intravenous anesthesia with propofol (10-40mg/kg/h) and fentanyl (10μg/kg/h). The rat mesentery was prepared for evaluation of macromolecular leakage, leukocyte adhesion and venular wall shear rate in postcapillary venules using IVM. Following baseline IVM recording, GTS-21 (1mg/kg) was applied simultaneously with, 1h prior to and 1h after administration of lipopolysaccharide (LPS, 5mg/kg). Test substances (crystalloid solution, LPS, GTS-21) were administered as volume equivalent intravenous infusions over 5min in the respective treatment groups. The consecutive IVMs were performed at 60, 120 and 180min after the baseline IVM. The systemic inflammatory response was evaluated by measuring TNF-α levels after the 180min IVM.nnnRESULTSnMicrovascular permeability was significantly reduced in animals treated with GTS-21 simultaneously and 1h after induction of endotoxemia. Leukocyte adhesion, venular wall shear rate and TNF-α levels were not affected by GTS-21 treatment compared to the untreated endotoxemic animals.nnnCONCLUSIONnGTS-21 has a protective effect on microvascular barrier function during endotoxemia. Considering its anti-inflammatory efficacy and safety profile, its clinical use might prove beneficial for the treatment of capillary leakage in sepsis therapy.

Data on microcirculatory parameters of GTS- 21 treated rats assessed by intravital microscopy

This article contains animal experimental data associated with the research article entitled “GTS-21 reduces microvascular permeability during experimental endotoxemia” (Schmidt et al., 2017) [1] (supplementary datasets of baseline intravital microscopic measurements, baseline TNF-α levels and vital parameters of the evaluated experimental groups are provided). Beneficial anti-inflammatory effects of cholinergic mediators on microvascular inflammation have been demonstrated by intravital microscopic investigations (Schmidt et al., 2015) [2], therefore we evaluated the effect of the cholinergic mediator GTS-21 on microcirculatory alterations during endotoxemia [1]. The data regarding microcirculatory effects of GTS-21 treatment ((3-(2,4-Dimethoxybenzylidene)-anabaseine dihydrochloride; 1 mg/kg; i.v.) in non-endotoxemic animals are presented in this article.

Impact of human sepsis on CCCTC-binding factor associated monocyte transcriptional response of Major Histocompatibility Complex II components

Background Antigen presentation on monocyte surface to T-cells by Major Histocompatibility Complex, Class II (MHC-II) molecules is fundamental for pathogen recognition and efficient host response. Accordingly, loss of Major Histocompatibility Complex, Class II, DR (HLA-DR) surface expression indicates impaired monocyte functionality in patients suffering from sepsis-induced immunosuppression. Besides the impact of Class II Major Histocompatibility Complex Transactivator (CIITA) on MHC-II gene expression, X box-like (XL) sequences have been proposed as further regulatory elements. These elements are bound by the DNA-binding protein CCCTC-Binding Factor (CTCF), a superordinate modulator of gene transcription. Here, we hypothesized a differential interaction of CTCF with the MHC-II locus contributing to an altered monocyte response in immunocompromised septic patients. Methods We collected blood from six patients diagnosed with sepsis and six healthy controls. Flow cytometric analysis was used to identify sepsis-induced immune suppression, while inflammatory cytokine levels in blood were determined via ELISA. Isolation of CD14++ CD16—monocytes was followed by (i) RNA extraction for gene expression analysis and (ii) chromatin immunoprecipitation to assess the distribution of CTCF and chromatin modifications in selected MHC-II regions. Results Compared to healthy controls, CD14++ CD16—monocytes from septic patients with immune suppression displayed an increased binding of CTCF within the MHC-II locus combined with decreased transcription of CIITA gene. In detail, enhanced CTCF enrichment was detected on the intergenic sequence XL9 separating two subregions coding for MHC-II genes. Depending on the relative localisation to XL9, gene expression of both regions was differentially affected in patients with sepsis. Conclusion Our experiments demonstrate for the first time that differential CTCF binding at XL9 is accompanied by uncoupled MHC-II expression as well as transcriptional and epigenetic alterations of the MHC-II regulator CIITA in septic patients. Overall, our findings indicate a sepsis-induced enhancer blockade mediated by variation of CTCF at the intergenic sequence XL9 in altered monocytes during immunosuppression.

The Interplay of Notch Signaling and STAT3 in TLR-Activated Human Primary Monocytes

The highly conserved Notch signaling pathway essentially participates in immunity through regulation of developmental processes and immune cell activity. In the adaptive immune system, the impact of the Notch cascade in T and B differentiation is well studied. In contrast, the function, and regulation of Notch signaling in the myeloid lineage during infection is poorly understood. Here we show that TLR signaling, triggered through LPS stimulation or in vitro infection with various Gram-negative and -positive bacteria, stimulates Notch receptor ligand Delta-like 1 (DLL1) expression and Notch signaling in human blood-derived monocytes. TLR activation induces DLL1 indirectly, through stimulated cytokine expression and autocrine cytokine receptor-mediated signal transducer and activator of transcription 3 (STAT3). Furthermore, we reveal a positive feedback loop between Notch signaling and Janus kinase (JAK)/STAT3 pathway during in vitro infection that involves Notch-boosted IL-6. Inhibition of Notch signaling by γ-secretase inhibitor DAPT impairs TLR4-stimulated accumulation of NF-κB subunits p65 in the nucleus and subsequently reduces LPS- and infection-mediated IL-6 production. The reduced IL-6 release correlates with a diminished STAT3 phosphorylation and reduced expression of STAT3-dependent target gene programmed death-ligand 1 (PD-L1). Corroborating recombinant soluble DLL1 and Notch activator oxaliplatin stimulate STAT3 phosphorylation and expression of immune-suppressive PD-L1. Therefore we propose a bidirectional interaction between Notch signaling and STAT3 that stabilizes activation of the transcription factor and supports STAT3-dependent remodeling of myeloid cells toward an immuno-suppressive phenotype. In summary, the study provides new insights into the complex network of Notch regulation in myeloid cells during in vitro infection. Moreover, it points to a participation of Notch in stabilizing TLR-mediated STAT3 activation and STAT3-mediated modulation of myeloid functional phenotype through induction of immune-suppressive PD-L1.

Advanced glycation endproducts induce self- and cross-tolerance in monocytes

IntroductionAdvanced glycation endproducts (AGEs) are well-known inflammatory mediators, which are recognized by immune cells through their corresponding receptor RAGE and have been shown to participate in the pathophysiology of a variety of acute as well as chronic inflammatory diseases. Nevertheless, no data are available on the aftermath of AGE recognition on immune cells.Materials and methodsWe used the monocytic cell line MonoMac6 as well as primary human monocytes for double stimulation experiments. We measured secreted as well as intracellular levels of TNF-α using ELISA and flow cytometry. In addition, gene expression of surface receptors (RAGE and TLR4) and TNF were measured by qPCR.ResultsStimulation with AGE leads to a dose-dependent induction of self- and cross-tolerance in both primary monocytes as well as the MonoMac6 cell line. The AGE tolerance depended neither on a decreased expression of RAGE or TLR4, nor on a decrease of TNF-α expression. Nevertheless, intracellular TNF-α was decreased, hinting towards a posttranscriptional regulation.ConclusionHigh levels of AGEs are capable to activate immune cellsxa0at first, but induce a secondary state of hypo-responsiveness in these cells. Based on the origin of its causal agent, we propose this phenomenon to be “metabolic tolerance”.

High-mobility group AT-hook protein 2 expression and its prognostic significance in MGMT methylated and unmethylated glioblastoma

High-mobility group AT-hook protein 2 (HMGA 2) is a transcription factor associated with malignancy and poor prognosis in a variety of human cancers. We correlated HMGA 2 expression with clinical parameters, survival, and O-6-methylguanine-DNA methyltransferase methylation status (MGMT) in glioblastoma patients. HMGA 2 expression was determined by performing quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry (IHC) in 44 glioblastoma patients and 5 non-tumorous brain specimens as controls. Gene expression levels of MGMT methylated vs. unmethylated patients, and gene expression levels between patient groups, both for qPCR and IHC data were compared using the Mann-Whitney U test. The relationship between HMGA 2 expression, progression-free survival and overall survival was analyzed using the Kaplan-Meier method and the log-rank test. P-values of <0.05 were considered statistically significant throughout the analyses. The mean age of patients at diagnosis was 57.4 ± 15.7 years, and the median survival was 16 months (SE 2.8; 95% CI, 10.6-21.4). HMGA 2 gene expression was significantly higher in glioblastoma compared to normal brain tissue on qPCR (mean, 0.35; SD, 0.27 vs. 0.03, SD, 0.05) and IHC levels (IRS mean, 17.21; SD, 7.43 vs. 3.20; SD, 1.68) (p=0.001). Survival analysis revealed that HMGA 2 overexpression was associated with a shorter progression-free and overall survival time in patients with methylation (n=24). The present study shows a tendency that HMGA 2 overexpression correlates with a poor prognosis of glioblastoma patients independent of MGMT methylation status. The results suggest that HMGA 2 could play an important role in the treatment of glioblastoma and could have a function in prognosis of this type of cancer.