Johannes Poeschl

RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(2)-integrins, we studied RAGE(-/-), Icam1(-/-), and RAGE(-/-) Icam1(-/-) mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.

Spleen tyrosine kinase Syk is critical for sustained leukocyte adhesion during inflammation in vivo

BackgroundDuring inflammation, β2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk.ResultsWe investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk-/- hematopoietic system. In unstimulated venules, Syk-/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk-/- and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk-/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk-/- leukocytes. Additional in vitro experiments revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised β2-integrin-mediated outside-in signaling.ConclusionWe provide substantial evidence for an important role of Syk in mediating β2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo.

Monitoring of intraportal liver cell application in children.

Despite recent advances and promising results in children, liver cell transplantation (LCT) should still be regarded as an experimental therapy. Several substantial complications are known from animal studies and individual patients. However, safety data on liver cell infusion in children are scarce. We used LCT in four children of different ages (3 weeks to 11 years, 3–40 kg) and underlying diseases [acute liver failure (n = 1), urea cycle disorders (n = 2), and Crigler-Najjar syndrome (n = 1)]. Vital parameters, portal vein flow (PVF), portal vein pressure (PVP), and liver enzymes were measured every 5 min during cell application and hourly thereafter between applications. An application protocol with discontinuation rules depending on changes in PVF and PVP was developed and successfully applied. Application was feasible in all children despite the catastrophic overall condition of the patient with acute liver failure. No application-related changes in vital parameters were found, and none of the children experienced clinical signs of portal vein thrombosis, pulmonary embolism, or anaphylactic reactions. Time courses for changes in PVF, PVP, and liver enzymes were obtained. Thorough monitoring of portal vein pressure and duplex sonography according to a defined protocol is likely to increase safety of cell application in pediatric LCT.

CD8+/FOXP3+-ratio in osteosarcoma microenvironment separates survivors from non-survivors: A multicenter validated retrospective study

Osteosarcoma is the most common primary bone tumor characterized by juvenile onset, tumor heterogeneity, and early pulmonary metastasis. Therapeutic improvement stagnates since more than two decades. Unlike major malignancies, biomarkers as prognostic factors at time of diagnosis are missing. Disease rareness hampers study recruitment of patient numbers sufficient to outweigh tumor heterogeneity. Here, we analyzed in a multicenter cohort the osteosarcoma microenvironment to reduce effects of tumor cell heterogeneity. We hypothesized that quantitative ratios of intratumoral CD8+T-cells to FOXP3+T-cells (CD8+/FOXP3+-ratios) provide strong prognostic information when analyzed by whole-slide imaging in diagnostic biopsies. We followed recommendations-for-tumor-marker-prognostic-studies (REMARK). From 150 included cases, patients with complete treatment were identified and assigned to the discovery (diagnosis before 2004) or the validation cohort (diagnosis 2004–2012). Highly standardized immunohistochemistry of CD8+ and FOXP3+, which was validated by methylation-specific gene analysis, was performed followed by whole-slide analysis and clinical outcome correlations. We observed improved estimated survival in patients with CD8+/FOXP3+-ratios above the median (3.08) compared to patients with lower CD8+/FOXP3+-ratios (p = 0.000001). No patients with a CD8+/FOXP3+-ratio above the third quartile died within the observation period (median follow-up 69 mo). Multivariate analysis demonstrated independence from current prognostic factors including metastasis and response to neoadjuvant chemotherapy. Data from an independent validation cohort confirmed improved survival (p = 0.001) in patients with CD8+/FOXP3+-ratios above 3.08. Multivariate analysis proofed that this observation was also independent from prognostic factors at diagnosis within the validation cohort. Intratumoral CD8+/FOXP3+-ratio in pretreatment biopsies separates patients with prolonged survival from non-survivors in osteosarcoma.

RAGE and ICAM-1 differentially control leukocyte recruitment during acute inflammation in a stimulus-dependent manner.

BackgroundThe receptor for advanced glycation endproducts, RAGE, is involved in the pathogenesis of many inflammatory conditions, which is mostly related to its strong activation of NF-κB but also due to its function as ligand for the β2-integrin Mac-1. To further dissect the stimulus-dependent role of RAGE on leukocyte recruitment during inflammation, we investigated β2-integrin-dependent leukocyte adhesion in RAGE-/- and Icam1-/- mice in different cremaster muscle models of inflammation using intravital microscopy.ResultsWe demonstrate that RAGE, but not ICAM-1 substantially contributes to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukocyte adhesion in TNF-α-pretreated cremaster muscle venules in a Mac-1-dependent manner. In contrast, fMLP-stimulated leukocyte adhesion in unstimulated cremaster muscle venules is independent of RAGE, but dependent on ICAM-1 and its interaction with LFA-1. Furthermore, chemokine CXCL1-stimulated leukocyte adhesion in surgically prepared cremaster muscle venules was independent of RAGE but strongly dependent on ICAM-1 and LFA-1 suggesting a differential and stimulus-dependent regulation of leukocyte adhesion during inflammation in vivo.ConclusionOur results demonstrate that RAGE and ICAM-1 differentially regulate leukocyte adhesion in vivo in a stimulus-dependent manner.

LPS- and LTA-Induced Expression of IL-6 and TNF-α in Neonatal and Adult Blood: Role of MAPKs and NF-κB

As nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) seem to be critical mediators in the inflammatory response, we studied the effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on (a) the activation of NF-κB and MAPKs and (b) the expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) with or without the specific inhibitors of these intracellular signal transduction pathways in neonatal cord and adult blood. TNF-α and IL-6 concentrations showed a sharp increase in the supernatants of cord and adult whole blood after stimulation. TNF-α concentrations were significantly higher, whereas IL-6 concentrations were tendentially lower in adult blood after stimulation. Stimulation with LPS or LTA resulted in a significantly decreased activation of p38 MAPK in neonatal compared with adult blood. Although LTA failed to induce additional ERK1/2 phosphorylation, LPS stimulation mediated the moderately increased levels of activated ERK1/2 in neonatal monocytes. The addition of the p38 MAPK inhibitor SB202190 significantly decreased IL-6 and TNF-α production upon LPS or LTA stimulation. Furthermore, the inhibition of ERK1/2 was able to reduce LPS-stimulated TNF-α production in neonatal blood. We conclude that p38 MAPK as well as ERK1/2 phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.

Energy expenditure and plasma catecholamines in preterm infants with mild chronic lung disease

The present study examined the hypothesis that the energy expenditure (EE) increases during the development of chronic lung disease (CLD) together with serum catecholamines as indicator of stress. Sixteen spontaneously breathing infants with gestational age of 28-34 weeks and birth weight of 870-1920 g were studied. Eight patients were at risk for CLD, eight were healthy controls. Measurements of indirect calorimetry were done weekly at postnatal ages of 2, 3, 4 and 5 weeks. Serum concentrations of adrenaline and noradrenaline were measured by means of a high-pressure liquid chromatography (HPLC) method. The eight CLD risk infants developed mild CLD with FiO(2) of 0.27-0.31 and characteristic radiographic signs at 28 days. Compared to the healthy controls, preterm infants with mild CLD showed increases in EE from week 3 (+67%) to week 5 (+46%). Plasma noradrenaline was increased significantly in the CLD infants when compared to the controls at week 3 (0.7+/-0.3 vs. 0.5+/-0.1 ng/ml; P<0.05) and more pronounced at week 4 (1.4+/-0.2 vs. 0.6+/-0.2 ng/ml; P<0.001) and 5 (1.1+/-0.3 vs. 0.7+/-0.2 ng/ml; P<0.01). Plasma adrenaline was markedly higher in the CLD risk group (mean overall value: 0.64+/-0.1 ng/ml) than in the controls (<0.1 ng/ml in all controls) from week 2 to 5. Regression analysis for the combined values of the infants with and without CLD showed that EE was directly correlated with heart rate, noradrenaline and adrenaline concentration at each of the four study weeks and with respiratory rate at weeks 2 and 3. Increased plasma catecholamine concentrations in preterm infants with CLD suggest that these infants experienced marked stress during the early stages of the disease. Increased EE may in part be a result of this stress.

Protein C Concentrate Controls Leukocyte Recruitment during Inflammation and Improves Survival during Endotoxemia after Efficient in Vivo Activation

Anti-inflammatory properties of protein C (PC) concentrate are poorly studied compared to activated protein C, although PC is suggested to be safer in clinical use. We investigated how PC interferes with the leukocyte recruitment cascade during acute inflammation and its efficacy during murine endotoxemia. We found that similar to activated protein infusion, intravenous PC application reduced leukocyte recruitment in inflamed tissues in a dose- and time-dependent manner. During both tumor necrosis factor-α induced and trauma-induced inflammation of the cremaster muscle, intravital microscopy revealed that leukocyte adhesion and transmigration, but not rolling, were profoundly inhibited by 100 U/kg PC. Moreover, PC blocked leukocyte emigration into the bronchoalveolar space during lipopolysaccharide (LPS) induced acute lung injury. PC was efficiently activated in a murine endotoxemia model, which reduced leukocyte infiltration of organs and strongly improved survival (75% versus 25% of control mice). Dependent on the inflammatory model, PC provoked a significant inhibition of leukocyte recruitment as early as 1 hour after administration. PC-induced inhibition of leukocyte recruitment during acute inflammation critically involves thrombomodulin-mediated PC activation, subsequent endothelial PC receptor and protease-activated receptor-1-dependent signaling, and down-regulation of intercellular adhesion molecule 1 leading to reduced endothelial inflammatory response. We conclude that during acute inflammation and sepsis, PC is a fast acting and effective therapeutic approach to block leukocyte recruitment and improve survival.

Feasibility of adjunct therapeutic hypothermia treatment for hyperammonemia and encephalopathy due to urea cycle disorders and organic acidemias

BACKGROUND Children with urea cycle disorders (UCDs) or organic acidemias (OAs) and acute hyperammonemia and encephalopathy are at great risk for neurological injury, developmental delay, intellectual disability, and death. Nutritional support, intravenous alternative pathway therapy, and dialysis are used to treat severe hyperammonemia associated with UCDs and nutritional support and dialysis are used to treat severe hyperammonemia in OAs. Brain protective treatment while therapy is initiated may improve neurological and cognitive function for the lifetime of the child. Animal experiments and small clinical trials in hepatic encephalopathy caused by acute liver failure suggest that therapeutic hypothermia provides neuroprotection in hyperammonemia associated encephalopathy. We report results of an ongoing pilot study that assesses if whole body cooling during rescue treatment of neonates with acute hyperammonemia and encephalopathy is feasible and can be conducted safely. METHODS Adjunct whole body therapeutic hypothermia was conducted in addition to standard treatment in acutely encephalopathic, hyperammonemic neonates with UCDs and OAs requiring dialysis. Therapeutic hypothermia was initiated using cooling blankets as preparations for dialysis were underway. Similar to standard therapeutic hypothermia treatment for neonatal hypoxic ischemic encephalopathy, patients were maintained at 33.5°C±1°C for 72h, they were then slowly rewarmed by 0.5°C every 3h over 18h. In addition data of age-matched historic controls were collected for comparison. RESULTS Seven patients were cooled using the pilot study protocol and data of seven historic controls were reviewed. All seven patients survived the initial rescue and cooling treatment, 6 patients were discharged home 2-4weeks after hospitalization, five of them feeding orally. The main complication observed in a majority of patients was hypotension. CONCLUSION Adjunct therapeutic hypothermia for neonates with UCDs and OAs receiving standard treatment was feasible and could be conducted safely in pediatric and neonatal intensive care units experienced in the application of therapeutic hypothermia in critically ill neonates. However, including adjunct therapeutic hypothermia in the already involved treatment regimen of critically ill patients with hyperammonemia and encephalopathy adds to the complexity of care and should not be done unless it is proven efficacious in a randomized clinical trial.

Effects of intralipid infusion on blood viscosity and other haemorheological parameters in neonates and children.

Aim: To study acute haemorheological effects of intralipid in preterm and full‐term neonates and children. Circulatory complications of intralipid infusion, such as increases in pulmonary and peripheral flow resistance, have been associated with impaired blood rheology. Methods: During total parenteral nutrition, 10 preterm infants, 10 full‐term neonates and 10 children received an initial dose of intralipid as continuous infusion (0.6 g/kg) over 4 h. Additionally, blood of 10 healthy preterm infants, 10 full‐term neonates and 10 adults was incubated with intralipid. Whole blood and plasma viscosity (capillary viscometer), red blood cell (RBC) deformability (rheoscope) and RBC aggregation (Myrenne aggregometer) were measured before and after intralipid infusion and before and after in vitro incubation of blood with intralipid. Results: During intralipid infusion, plasma triglyceride levels increased from 0.13 ± 0.27 to 2.16 ± 0.68g/l in the preterm infants, from 0.14 ± 0.21 to 1.64 ± 0.54g/l in the full‐term neonates and from 0.65 ± 0.31 to 2.26 ± 0.60 g/l in the children. Whole blood viscosity decreased by about 10% after intralipid in all three groups due to similar decreases in haematocrit. RBC aggregation decreased by about 20% after intralipid infusion. Plasma proteins, plasma viscosity and RBC deformation were not affected by intralipid. In vitro incubation of blood with intralipid resulted in a marked reduction of RBC aggregation that was related to the intralipid concentration. At intralipid concentrations of 4 and 8 mg/ml, no RBC aggregation was noted in preterm and full‐term neonates. In adults, RBC aggregation decreased by 50%.