Benjamin Funke

High-Mobility Group Box-1 in Ischemia-Reperfusion Injury of the Heart

Background— High-mobility group box-1 (HMGB1) is a nuclear factor released by necrotic cells and by activated immune cells. HMGB1 signals via members of the toll-like receptor family and the receptor for advanced glycation end products (RAGE). Although HMGB1 has been implicated in ischemia/reperfusion (I/R) injury of the liver and lung, its role in I/R injury of the heart remains unclear. Methods and Results— Here, we demonstrate that HMGB1 acts as an early mediator of inflammation and organ damage in I/R injury of the heart. HMGB1 levels were already elevated 30 minutes after hypoxia in vitro and in ischemic injury of the heart in vivo. Treatment of mice with recombinant HMGB1 worsened I/R injury, whereas treatment with HMGB1 box A significantly reduced infarct size and markers of tissue damage. In addition, HMGB1 inhibition with recombinant HMGB1 box A suggested an involvement of the mitogen-activated protein kinases jun N-terminal kinase and extracellular signal-regulated kinase 1/2, as well as the nuclear transcription factor nuclear factor-&kgr;B in I/R injury. Interestingly, infarct size and markers of tissue damage were not affected by administration of recombinant HMGB1 or HMGB1 antagonists in RAGE−/− mice, which demonstrated significantly reduced damage in reperfused hearts compared with wild-type mice. Coincubation studies using recombinant HMGB1 in vitro induced an inflammatory response in isolated macrophages from wild-type mice but not in macrophages from RAGE−/− mice. Conclusions— HMGB1 plays a major role in the early event of I/R injury by binding to RAGE, resulting in the activation of proinflammatory pathways and enhanced myocardial injury. Therefore, blockage of HMGB1 might represent a novel therapeutic strategy in I/R injury.

Sepsis and Major Abdominal Surgery Lead to Flaking of the Endothelial Glycocalix

BACKGROUND Recent evidence suggests that the endothelial glycocalix plays an important role in lethal outcomes following sepsis. We therefore tested if the endothelial glycocalix is shed in patients with sepsis compared with patients after major abdominal surgery and healthy volunteers. MATERIAL AND METHODS A total of 150 individuals were tested for levels of inflammatory markers (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1], interleukin-6 [IL-6]) and glycocalix markers (syndecan-1, heparan sulfate). Three groups consisted of patients with severe sepsis or septic shock, patients after major abdominal surgery without systemic inflammatory response syndrome, and healthy volunteers. Blood was drawn, at the time of diagnosis or surgery, and 6, 24, and 48h later. We correlated these markers to each other and to clinically used inflammation markers. RESULTS Levels of inflammatory markers were markedly higher in patients with sepsis compared with patients after major abdominal surgery and healthy volunteers. After major abdominal surgery, glycocalix markers in human plasma were at levels comparable to patients with sepsis. In patients with sepsis, levels of IL-6 correlated with syndecan-1, ICAM-1, VCAM-1, and lactate, while ICAM-1 furthermore correlated with CRP and lactate levels. CONCLUSION High levels of glycocalix markers indicated that significant flaking of the endothelial glycocalix occurred in patients with sepsis, and to a lesser extent in patients after major abdominal surgery. This novel finding could explain the nonspecific capillary leaking syndrome of patients with sepsis and after major abdominal surgery, and may identify new targets for treating those patient populations.

Functional characterisation of decoy receptor 3 in Crohn's disease.

Aims: Both epithelial barrier dysfunction and apoptosis resistance of immune cells contribute to the pathogenesis of Crohn’s disease. The soluble decoy receptor 3 (DcR3) acts in an anti-apoptotic manner by neutralising the death ligand CD95L. Here, we investigated the possible involvement of DcR3 in Crohn’s disease. Methods: The epithelial fraction of human small intestinal mucosa samples was obtained by laser microdissection. Expression of DcR3 was examined by global gene expression profiling, quantitative reverse transcription polymerase chain reaction, immunoblot analysis, and immunohistochemistry. DcR3 concentrations in the serum of patients with Crohn’s disease were measured by enzyme-linked immunosorbent assay. Apoptosis assays were performed to study the effects of DcR3 in intestinal epithelial cells and lamina propria T cells. Results: DcR3 is over-expressed in the epithelial layer of ileum specimens in patients with Crohn’s disease, both at actively inflamed and non-active sites. DcR3 serum levels are significantly elevated in patients with active and non-active Crohn’s disease as compared to healthy controls. The expression of DcR3 in intestinal epithelial cells is induced by tumour necrosis factor α. Increased DcR3 expression is associated with activation of nuclear factor kappa B (NF-κB) and results in protection of intestinal epithelial cells and lamina propria T cells from CD95L-induced apoptosis. Conclusions: DcR3 may promote inflammation in Crohn’s disease by inhibiting CD95L-induced apoptosis of epithelial and immune cells as well as by inducing NF-κB activation.

Basal Caspase Activity Promotes Migration and Invasiveness in Glioblastoma Cells

Glioblastomas, the most malignant of all brain tumors, are characterized by cellular resistance to apoptosis and a highly invasive growth pattern. These factors contribute to the poor response of glioblastomas to radiochemotherapy and prevent their complete neurosurgical resection. However, the driving force behind the distinct motility of glioma cells is only partly understood. Here, we report that in the absence of cellular stress and proapoptotic stimuli, human glioblastoma cells exhibit a constitutive activation of caspases in vivo and in vitro. The inhibition of caspases by various peptide inhibitors decreases the migration of cells in scrape motility assays and the invasiveness of cells in spheroid assays. Similarly, specific small interfering RNA– or antisense-mediated down-regulation of caspase-3 and caspase-8 results in an inhibition of the migratory potential of glioma cells. The constitutive caspase-dependent motility of glioblastoma cells is independent of CD95 activation and it is not mediated by mitogen-activated protein/extracellular signal-regulated kinase kinase signaling. The basal caspase activity is accompanied by a constant cleavage of the motility-associated gelsolin protein, which may contribute to the caspase-mediated promotion of migration and invasiveness in glioblastoma cells. Our results suggest that the administration of low doses of caspase inhibitors that block glioma cell motility without affecting the execution of apoptotic cell death may be exploited as a novel strategy for the treatment of glioblastomas. (Mol Cancer Res 2007;5(12):1232–40)

Pathology of the large intestine in patients with vascular type Ehlers-Danlos syndrome

The vascular type of Ehlers-Danlos syndrome (type IV) is an infrequent disease caused by heterozygous germline mutations in the procollagen 3A gene (COL3A1). Clinical signs include characteristic facial features, easy bruising, and a translucent skin. These signs are less obvious than the hyperflexibility of skin and joints seen in other types of Ehlers-Danlos syndrome. Therefore, diagnosis of Ehlers-Danlos syndrome type IV is usually not considered until complications have occurred. Complications include spontaneous ruptures of vessels and hollow organs, particularly the colon. We, herein, report pathologic findings in colon specimens from related Ehlers-Danlos syndrome type IV patients. Thorough examination revealed abnormalities of the large bowel architecture including abrupt changes in the caliber of the lamina muscularis, secondary diverticula formation, and strongly reduced expression of abnormal collagen 3. These findings are not seen in other diseases of the colon and should prompt the pathologist to include Ehlers-Danlos syndrome type IV in the differential diagnosis of spontaneous bowel perforation in younger patients.

Tissue-Resident Macrophages Are Productively Infected Ex Vivo by Primary X4 Isolates of Human Immunodeficiency Virus Type 1

ABSTRACT Infection of macrophages has been implicated as a critical event in the transmission and persistence of human immunodeficiency virus type 1 (HIV-1). Here, we explore whether primary X4 HIV-1 isolates can productively infect tissue macrophages that have terminally differentiated in vivo. Using immunohistochemistry, HIV-1 RNA in situ hybridization, and confocal immunofluorescence microscopy, we demonstrate that macrophages residing in human tonsil blocks can be productively infected ex vivo by primary X4 HIV-1 isolates. This challenges the model in which macrophage tropism is a key determinant of the selective transmission of R5 HIV-1 strains. Infection of tissue macrophages by X4 HIV-1 may be highly relevant in vivo and contribute to key events in HIV-1 pathogenesis.

The adenosine deaminase inhibitor erythro-9-[2-hydroxyl-3-nonyl]-adenine decreases intestinal permeability and protects against experimental sepsis: a prospective, randomised laboratory investigation

IntroductionThe treatment of septic conditions in critically ill patients is still one of medicines major challenges. Cyclic nucleotides, adenosine and its receptors play a pivotal role in the regulation of inflammatory responses and in limiting inflammatory tissue destruction. The aim of this study was to verify the hypothesis that adenosine deaminase-1 and cyclic guanosine monophosphate-stimulated phosphodiesterase inhibition by erythro-9-[2-hydroxyl-3-nonyl]-adenine could be beneficial in experimental endotoxicosis/sepsis.MethodWe used two established animal models for endotoxicosis and sepsis. Twenty-four male Wistar rats that had been given intravenous endotoxin (Escherichia coli lipopolysaccharide) were treated with either erythro-9-[2-hydroxyl-3-nonyl]-adenine infusion or 0.9% saline during a study length of 120 minutes. Sepsis in 84 female C57BL/6 mice was induced by caecal ligation and puncture. Animals were treated with repeated erythro-9-[2-hydroxyl-3-nonyl]-adenine injections after 0, 12 and 24 hours or 4, 12 and 24 hours for delayed treatment.ResultsIn endotoxaemic rats, intestinal production of hypoxanthine increased from 9.8 +/- 90.2 μmol/l at baseline to 411.4 +/- 124.6 μmol/l and uric acid formation increased from 1.5 +/- 2.3 mmol/l to 13.1 +/- 2.7 mmol/l after 120 minutes. In endotoxaemic animals treated with erythro-9-[2-hydroxyl-3-nonyl]-adenine, we found no elevation of adenosine metabolites. The lactulose/L-rhamnose ratio (14.3 versus 4.2 in control animals; p = 2.5 × 10-7) reflects a highly permeable small intestine and through the application of erythro-9-[2-hydroxyl-3-nonyl]-adenine, intestinal permeability could be re-established. The lipopolysaccharide animals had decreased L-rhamnose/3-O-methyl-D-glucose urine excretion ratios. Erythro-9-[2-hydroxyl-3-nonyl]-adenine reduced this effect. The mucosa damage score of the septic animals was higher compared with control and therapy animals (p < 0.05). Septic shock induction by caecal ligation and puncture resulted in a 160-hour survival rate of about 25%. In contrast, direct adenosine deaminase-1 inhibition resulted in a survival rate of about 75% (p = 0.0018). A protective effect was still present when erythro-9-[2-hydroxyl-3-nonyl]-adenine treatment was delayed for four hours (55%, p = 0.029).ConclusionsWe present further evidence of the beneficial effects achieved by administering erythro-9-[2-hydroxyl-3-nonyl]-adenine, an adenosine deaminase-1 and cyclic guanosine monophosphate-stimulated phosphodiesterase inhibitor, in an endotoxicosis and sepsis animal model. This suggests a potential therapeutic option in the treatment of septic conditions.

Up-regulation of the phosphoinositide 3-kinase pathway in human lamina propria T lymphocytes.

Human intestinal lamina propria T lymphocytes (LPT), when investigated ex vivo, exhibit functional properties profoundly different from those of peripheral blood T lymphocytes (PBT). One prominent feature represents their enhanced sensitivity to CD2 stimulation when compared to PBT. Given that LPT are hyporesponsive to T cell receptor (TCR)/CD3 stimulation, an alternative activation mode, as mimicked by CD2 triggering in vitro, may be functional in mucosal inflammation in vivo. This study provides insight into signalling events associated with the high CD2 responsiveness of LPT. When compared to PBT, LPT show an increased activation of the phosphoinositide 3/protein kinase B/glycogen synthase kinase 3β (PI3‐kinase/AKT/GSK‐3β) pathway in response to CD2 stimulation. Evidence is provided that up‐regulation of this pathway contributes to the enhanced CD2‐induced cytokine production in LPT. Given the importance of TCR‐independent stimulation for the initiation of intestinal immune responses analysis of signalling pathways induced by ‘co‐stimulatory’ receptors may provide valuable information for therapeutic drug design.

Fragment reconstruction of coronary arteries using transesophageal echocardiography for coronary diagnostics

AIMS Ultrasound differs procedurally from the established methods for non-invasive coronary visualization and is therefore an interesting alternative for non-invasive diagnostics. In this study, fragment reconstruction of coronary arteries by transesophageal echocardiography (FRC-TEE) was investigated for the first time in a patient population being evaluated for coronary angiography. METHODS AND RESULTS Ultrasonic and angiographic findings were compared visually and using quantitative measurements in 50 patients. One hundred and seventy-one vessels were examined by FRC-TEE. The total lengths visualized were 9.6 +/- 1.7 cm for the right coronary artery, 7.0 +/- 1.1 cm for left circumflex, 3.9 +/- 1.2 cm for left anterior descending (LAD), and 1.5 +/- 0.8 cm for the left main coronary artery. There was high concordance between results of both procedures. Sixty-three stenoses were detected using FRC-TEE. The mean difference in degree of stenosis between techniques was 0.2 +/- 5.1%. Stents could be visualized in 19 segments. FRC-TEE detected distal stenoses of the coronary arteries to only a limited extent: 14 stenoses and 2 stents, predominantly in the LAD artery (n = 13), were not identified. CONCLUSIONS FRC-TEE is a potential method for diagnosing coronary artery disease. FRC-TEE and angiography yield comparable findings during the evaluation of coronary lesions. Further investigations are needed to verify the encouraging findings and define FRC-TEEs applications.