Matthias Zimmermann

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.

Secretome of peripheral blood mononuclear cells enhances wound healing.

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SECPBMC). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SECPBMC containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SECPBMC on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SECPBMC containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SECPBMC induced proliferation and tube-formation in a matrigel-assay. In addition, SECPBMC treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.

Analysis of the Secretome of Apoptotic Peripheral Blood Mononuclear Cells: Impact of Released Proteins and Exosomes for Tissue Regeneration

We previously showed that, when peripheral blood mononuclear cells (PBMCs) were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. This study aimed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. Bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes were primarily involved in (a) pro-angiogenic and regenerative pathways and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects. We tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed chest, acute myocardial infarction. We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes. Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs. In addition our findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine.

Comparison of plasma concentrations of soluble ST2 measured by three different commercially available assays: The MBL ST2 assay, the Presage ST2 assay, and the R&D ST2 assay

The protein ST2 is a member of the interleukin-1 receptor family [1]. Blood concentrations of the soluble isoform of ST2 (sST2) are increased in inflammatory diseases and in heart disease and are considered a prognostic marker in both [1–5]. In published studies, mainly three different assays for determination of circulating sST2 concentrations have been used, the MBL assay, the R&D assay, and more recently the Presage assay. The latter is FDA cleared and CE marked, while the other twomethods are research assays. It is, however, currently unclear whether sST2 concentrations obtained by these threemethods are comparable. Here we report the results of a method comparison study in order to generate data on this important issue. The aim of the study was to compare sST2 plasma concentrations as measured by three commercially available assays, namely the MBL ST2 ELISAKit, code-number 7638 (Medical & Biological Laboratories,Woburn, MA, USA), the Presage ST2 assay (Critical Diagnostics, San Diego, CA, USA), and R&D DuoSet ELISA human ST2/IL-1 R4, catalog-number DY523 (R&D Systems, Minneapolis, MN, USA). For this comparison study, we used plasma samples from 45 male patients with a variety of diseases recruited in the St. John of God Hospital in Linz, Austria. An EDTA anticoagulated blood sample was collected for quantification of sST2. Measurement of sST2 with the three assays was performed according to the package inserts and as previously described [6–8]. For all three assays, sST2 measurements of each plasma sample were done in duplicate on the same microwell plates, and the mean of these two determinations was used for further analyses. The study protocol was approved by the local ethics committee in accordance with the Declaration of Helsinki, and all study participants gave informed consent. The 45 study participants had sST2 plasma concentrations between the highest and the lowest standards of the three assays. This prerequisite was set to evaluate plasma concentrations within the measurement ranges of the assays and to avoid problems with assay detection limits in case of very low plasma concentrations. We had thus a measurement range of 0.1875–12 ng/mL for the MBL ST2 assay (based on a 3-fold dilution of patient samples), a measurement

Bone-Conditioned Medium Changes Gene Expression in Bone-Derived Fibroblasts.

PURPOSE Autologous bone is used for augmentation in the course of oral implant placement. Bone grafts release paracrine signals that can modulate mesenchymal cell differentiation in vitro. The detailed genetic response of the bone-derived fibroblasts to these paracrine signals has remained elusive. Paracrine signals accumulate in bone-conditioned medium (BCM) prepared from porcine cortical bone chips. MATERIALS AND METHODS In this study, bone-derived fibroblasts were exposed to BCM followed by a whole genome expression profiling and downstream quantitative reverse transciptase polymerase chain reaction of the most strongly regulated genes. RESULTS The data show that ADM, IL11, IL33, NOX4, PRG4, and PTX3 were differentially expressed in response to BCM in bone-derived fibroblasts. The transforming growth factor beta (TGF-β) receptor 1 antagonist SB431542 blocked the effect of BCM on the expression of the gene panel, except for IL33. CONCLUSION These in vitro results extend existing evidence that cortical bone chips release paracrine signals that provoke a robust genetic response in mesenchymal cells that is not exclusively mediated via the TGF-β receptor. The present data provide further insights into the process of graft consolidation.

Mononuclear cell secretome protects from experimental autoimmune myocarditis

Abstract Aims Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. Methods and results BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. Conclusion MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases.

Secretome of apoptotic peripheral blood cells (APOSEC) attenuates microvascular obstruction in a porcine closed chest reperfused acute myocardial infarction model: role of platelet aggregation and vasodilation.

Although epicardial blood flow can be restored by an early intervention in most cases, a lack of adequate reperfusion at the microvascular level is often a limiting prognostic factor of acute myocardial infarction (AMI). Our group has recently found that paracrine factors secreted from apoptotic peripheral blood mononuclear cells (APOSEC) attenuate the extent of myocardial injury. The aim of this study was to determine the influence of APOSEC on microvascular obstruction (MVO) in a porcine AMI model. A single dose of APOSEC was intravenously injected in a closed chest reperfused infarction model. MVO was determined by magnetic resonance imaging and cardiac catheterization. Role of platelet function and vasodilation were monitored by means of ELISA, flow cytometry, aggregometry, western blot and myographic experiments in vitro and in vivo. Treatment of AMI with APOSEC resulted in a significant reduction of MVO. Platelet activation markers were reduced in plasma samples obtained during AMI, suggesting an anti-aggregatory capacity of APOSEC. This finding was confirmed by in vitro tests showing that activation and aggregation of both porcine and human platelets were significantly impaired by co-incubation with APOSEC, paralleled by vasodilator-stimulated phosphoprotein (VASP)-mediated inhibition of platelets. In addition, APOSEC evidenced a significant vasodilatory capacity on coronary arteries via p-eNOS and iNOS activation. Our data give first evidence that APOSEC reduces the extent of MVO during AMI, and suggest that modulation of platelet activation and vasodilation in the initial phase after myocardial infarction contributes to the improved long-term outcome in APOSEC treated animals.

Increased serum levels of HSP27 as a marker for incipient chronic obstructive pulmonary disease in young smokers

Background: Although chronic obstructive pulmonary disease (COPD) is amongst the leading causes of morbidity and mortality, no biomarkers for its early detection are known. We have recently demonstrated that COPD is accompanied by elevated serum heat shock protein (HSP) 27 levels as compared to a control population. Objectives: In an open prospective study, we investigated whether elevated HSP27 levels are associated with the early radiological signs of COPD, i.e. air trapping (AT), emphysema (E) and impaired lung function. Methods: In total, 120 apparently healthy smokers underwent lung function testing and serum sampling. Serum levels of HSP27, phospho-HSP27, CXCR2 chemokines and proteins related to inflammation, tissue remodeling and apoptosis were evaluated by ELISA. Of these 120 subjects, 94 voluntarily underwent a high-resolution computed tomography scan. Results: AT or AT and E were detected in 57.45%. Subjects with AT and E (n = 23) showed significantly higher HSP27 levels than those without any pathology [i.e. nothing abnormal detected (NAD)] (4,618 ± 1,677 vs. 3,282 ± 1,607 pg/ml; p = 0.0081). In a univariate logistic regression model including NAD and AT and E, the area under the curve of HSP27 in the receiver-operating-characteristic curve was 0.724, (0.594–0.854, 95% CI; p = 0.0033). Interestingly, proinflammatory IL-8 was elevated in those subjects with evidence of AT and E compared to those with AT and NAD. Lung function did not correlate with increased HSP27 levels or pathological radiological findings. Conclusions: HSP27 serum levels correlated with the early radiological signs of COPD, whereas lung function did not match with radiological findings or HSP27 serum levels. Serum HSP27 levels may serve as a potential marker to identify the early signs of COPD independent of lung function in young smokers.

Stromal Expression of Heat-Shock Protein 27 Is Associated with Worse Clinical Outcome in Patients with Colorectal Cancer Lung Metastases

Background Pulmonary metastases are common in patients with primary colorectal cancer (CRC). Heat-shock protein 27 (Hsp27) is upregulated in activated fibroblasts during wound healing and systemically elevated in various diseases. Cancer-associated fibroblasts (CAFs) are also thought to play a role as prognostic and predictive markers in various malignancies including CRC. Surprisingly, the expression of Hsp27 has never been assessed in CAFs. Therefore we aimed to investigate the expression level of Hsp27 in CAFs and its clinical implications in patients with CRC lung metastases. Methods FFPE tissue samples from 51 pulmonary metastases (PMs) and 33 paired primary tumors were evaluated for alpha-SMA, CD31, Hsp27 and vimentin expression by immunohistochemistry and correlated with clinicopathological variables. 25 liver metastases served as control group. Moreover, serum samples (n=10) before and after pulmonary metastasectomy were assessed for circulating phospho-Hsp27 and total Hsp27 by ELISA. Results Stromal expression of Hsp27 was observed in all PM and showed strong correlation with alpha-SMA (P<0.001) and vimentin (P<0.001). Strong stromal Hsp27 was associated with higher microvessel density in primary CRC and PM. Moreover, high stromal Hsp27 and αSMA expression were associated with decreased recurrence-free survival after pulmonary metastasectomy (P=0.018 and P=0.008, respectively) and overall survival (P=0.031 and P=0.017, respectively). Serum levels of phospho- and total Hsp27 dropped after metastasectomy to levels comparable to healthy controls. Conclusions Herein we describe for the first time that Hsp27 is highly expressed in tumor stroma of CRC. Stromal α-SMA and Hsp27 expressions correlate with the clinical outcome after pulmonary metastasectomy. Moreover, serum Hsp27 might pose a future marker for metastatic disease in CRC.

Long-acting beneficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction

The quantity of cells with paracrine effects for use in myocardial regeneration therapy is limited. This study investigated the effects of catheter-based endomyocardial delivery of secretome of 2.5 × 10(9) apoptotic peripheral blood mononuclear cells (APOSEC) on porcine chronic post-myocardial infarction (MI) left ventricular (LV) dysfunction and on gene expression. Closed-chest reperfused MI was induced in pigs by 90-min occlusion followed by reperfusion of the mid-LAD (day 0). At day 30, animals were randomized to receive porcine APOSEC (n = 8) or medium solution (control; n = 8) injected intramyocardially into the MI border zone using 3D NOGA guidance. At day 60, cardiac MRI with late enhancement and diagnostic NOGA (myocardial viability) were performed. Gene expression profiling of the infarct core, border zone, and normal myocardium was performed using microarray analysis and confirmed by quantitative real-time PCR. Injection of APOSEC significantly decreased infarct size (p < 0.05) and improved cardiac index and myocardial viability compared to controls. A trend towards higher LV ejection fraction was observed in APOSEC vs. controls (45.4 ± 5.9% vs. 37.4 ± 8.9%, p = 0.052). Transcriptome analysis revealed significant downregulation of caspase-1, tumor necrosis factor and other inflammatory genes in APOSEC-affected areas. rtPCR showed higher expression of myogenic factor Mefc2 (p < 0.05) and downregulated caspase genes (p < 0.05) in APOSEC-treated pigs. In conclusion, overexpression of MEF2c and repression of caspase was related to decreased infarct size and improved cardiac function in secretome-treated animals. Altered gene expression 1-month post-APOSEC treatment proved the long-acting effects of cell-free therapy with paracrine factors.