Konrad Hoetzenecker

ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression

Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2–related factor 2 (NRF2)–dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3−/− mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3−/− mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3−/−Il6−/− mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3−/− mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.

Heightened levels of circulating 20S proteasome in critically ill patients

Background  Recently, circulating proteasome core particles (20S proteasome) have been suggested as a marker of cell damage and immunological activity in autoimmune diseases. Aberrant leucocyte activation and increased lymphocyte apoptosis with consecutive T‐cell unresponsiveness is deemed to play a pivotal role in the sepsis syndrome. Moreover sepsis‐induced muscle proteolysis mainly reflects ubiqutin proteasome‐dependent protein degradation. We therefore sought to investigate serum levels of 20S proteasome in critical ill patients.

Primary sources and immunological prerequisites for sST2 secretion in humans

AIMS Serum levels of the soluble growth stimulation gene-2 (sST2) are elevated in heart and pulmonary diseases. However, the relationship of the sST2/interleukin (IL)-33 axis and its triggers as well as its organ distribution is still not known. This study was thus designed to investigate the cellular origin and regulation of sST2 and IL-33 in vitro and in vivo. METHODS AND RESULTS sST2 and IL-33 gene expression and protein secretion were analysed in pooled organ-specific cDNAs and in primary cell cultures, respectively, by RT-PCR and ELISA technology. The strongest sST2 mRNA expression was detected in heart and lung tissues, which correlated with spontaneous secretion of sST2 protein in vitro. The inflammatory cytokines IL-1alpha, IL-1beta, and tumour necrosis factor alpha as well as supernatants of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells led to an enhanced secretion of sST2 in cultured cardiac myocytes and lung alveolar epithelial cells. These cytokines enhanced sST2 secretion via an NFkappaB-dependent mechanism. In addition, LPS stimulation in humans in vivo induced a short-term inflammatory response that was followed by a massive enhancement of sST2 secretion. CONCLUSION These results identify the primary sources and inflammatory triggers for the enhancement of sST2 secretion and demonstrate a relationship between inflammation and the secretion of a bioactive member of the IL-1R family, both in vitro and in vivo.

T cell senescence and contraction of T cell repertoire diversity in patients with chronic obstructive pulmonary disease

Pathogenetic mechanisms leading to chronic obstructive pulmonary disease (COPD) remain poorly understood. Because clonogenic T cells (CD4+CD28null) were shown to be increased in autoimmune diseases we hypothesized that CD4+CD28null T cells play a role in COPD. Here we describe that enhanced presence of CD4+CD28null cells is associated with impaired lung function. Sixty‐four patients and controls were included. T cell phenotype was analysed using flow cytometry. Enzyme‐linked immunosorbent assays were utilized to determine cytokines. Statistical evaluations were performed using non‐parametric group comparisons and correlations. A logistic regression model was used to determine predictive values of CD4+CD28null in the diagnosis of COPD. Populations of CD4+ T cells lacking surface co‐stimulatory CD28 were enlarged significantly in evaluated patients when compared with controls. Natural killer (NK)‐like T cell receptors (CD94, 158) and intracellular perforin, granzyme B were increased in CD4+CD28null cells. Cytokine production after triggering of peripheral blood mononuclear cells (PBMCs) was elevated in patients at early disease stages. Receiver operating characteristic curve plotting revealed that presence of CD4+CD28null T cells has a diagnostic value. These CD4+CD28null T cells show increased expression of NK‐like T cell receptors (CD94, 158) and intracellular perforin and granzyme B. Furthermore, triggering of PBMCs obtained from patients with mild COPD led to increased interferon‐γ and tumour necrosis factor‐α production in vitro compared with controls. Our finding of increased CD4+CD28null T cells in COPD indicates that chronic antigen exposure, e.g. through contents of smoke, leads to loss of CD28 and up‐regulation of NK cell receptors expression on T cells in susceptible patients.

Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models

Anti‐angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti‐angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre‐existing vessels from surrounding tissue (vessel co‐option). As anti‐angiogenic therapies were designed to target only new blood vessel growth, vessel co‐option has been proposed as a mechanism that could drive resistance to anti‐angiogenic therapy. However, vessel co‐option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti‐angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co‐option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co‐opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co‐opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co‐option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co‐option mediates resistance to the anti‐angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co‐option in lung metastases occurs through at least three distinct mechanisms, that vessel co‐option occurs frequently in lung metastases, and that vessel co‐option could mediate resistance to anti‐angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co‐option are therefore warranted.

Thrombospondin-1: a unique marker to identify in vitro platelet activation when monitoring in vivo processes.

Summary.  Background:  Measuring platelet activation in patients has become a potent method to investigate pathophysiological processes. However, the commonly applied markers are sensitive to detrimental influences by in vitro platelet activation during blood analysis.

Irradiated cultured apoptotic peripheral blood mononuclear cells regenerate infarcted myocardium

Background  Acute myocardial infarction (AMI) is followed by post AMI cardiac remodelling, often leading to congestive heart failure. Homing of c‐kit+ endothelial progenitor cells (EPC) has been thought to be the optimal source for regenerating infarcted myocardium.

Discrimination of clinical stages in non-small cell lung cancer patients by serum HSP27 and HSP70: a multi-institutional case-control study.

INTRODUCTION Lung cancer represents a major healthcare problem. Accordingly, there is an urgent need to identify serum biomarkers for early diagnosis of lung pathology. We have recently described that patients with manifest COPD evidence elevated levels of heat shock proteins (HSPs). Based on these data, we speculated whether HSPs are also increased in patients with diagnosed lung cancer. METHODS Serum levels of HSP27, phospho-HSP27 (pHSP27) and HSP70 in patients with non-small cell lung cancer (NSCLC) diagnosed at an early (stages I-II, n=37) or advanced (stages IIIA-IV, n=72) stage were determined by using ELISA. Healthy smokers (n=24), healthy never-smoker volunteers (n=33) and COPD patients (n=34) according to GOLD classification served as control population. RESULTS Serum levels of HSP27 were elevated in patients with NSCLC diagnosed at an early or advanced stage when compared with both healthy control groups (P<0.005 and P<0.0001 respectively). Statistically significant differences were furthermore found between the groups of patients with early vs. advanced stage NSCLC (P=0.0021). Serum levels of HSP70 were also significantly elevated in patients with NSCLC diagnosed at an early or at an advanced stage when compared with either healthy control groups (P=0.0028 and P<0.0001 respectively). In univariate logistic regression models including healthy subjects and patients with NSCLC, HSP70 had an area under the curve (AUC) of 0.779 (P<0.0001) and HSP27 showed an AUC of 0.870 (P<0.0001). CONCLUSION Our data suggest that serum HSP27 levels might serve as a possible tool to discriminate between early and advanced stages NSCLC.

Alpha-Gal specific IgG immune response after implantation of bioprostheses.

BACKGROUND We have previously shown that the alpha-Gal (Galalpha1.3-Galbeta1-4GlcNAc-R) epitope is a relevant xenoantigen present on bioprostheses utilized in cardiac surgery and elicits an alpha-Gal specific IgM immune response. We sought to investigate whether that immune response continues after valve implantation. MATERIALS AND METHODS We collected plasma samples from patients who underwent bioprosthesis implantation (n = 19) or mechanical valve replacement (n = 8), respectively, prior to, at 10 days and at 3 months after cardiac surgery. ELISA was utilized to quantify alpha-Gal specific IgG and IgG subclasses. 3 bioprosthetic tissue samples were obtained from patients who had to undergo re-operation within 1 week (n = 1) or at 12-15 months (n = 2) after the initial operation. We utilized confocal laser scanning microscopy (CLSM) to detect the presence of alpha-Gal epitopes (IB4) and cell nuclei (DAPI). RESULTS alpha-Gal specific IgG was significantly increased 3 months after implantation of bioprostheses compared to preoperative values (p < 0.001) and was significantly higher than alpha-Gal specific IgG levels of the control group (p < 0.05). IgG3 was the major subclass directed against alpha-Gal (p < 0.05, pre- vs. postoperative values). In CLSM analysis we demonstrated that bioprostheses explanted 1 week after implantation contained IB4/DAPI positive cells within the collagen matrix. In contrast, in patients who underwent reoperation after 12 months, porcine tissue showed a complete lack of IB4/DAPI. CONCLUSION Our results indicate that the implantation of bioprostheses elicits a specific humoral immune response against alpha-Gal bearing cells compared to controls within 3 months after cardiac surgery. The complete absence of IB4/DAPI positive structures 12 months after implantation indicates a specific degradation of alpha-Gal bearing cells through previous exposure to the human blood circuit.

Increased soluble serum markers caspase-cleaved cytokeratin-18, histones, and ST2 indicate apoptotic turnover and chronic immune response in COPD.

Introduction: Chronic obstructive pulmonary disease (COPD) is a worldwide burden and a major cause of death. The disease is accompanied by chronic inflammation and increased cellular turnover that is partly due to an overwhelming induction of apoptosis. In this study, we hypothesized that systemic markers of apoptosis are altered in patients with mild‐to‐severe COPD.