January 2012 Paper accepted in Respiration. International Journal of Thoracic Medicine

Increased serum levels of HSP27 as a marker for incipient COPD in young smokers

Ankersmit HJ, Nickl S, Hoeltl E, Toepker M, Lambers C, Mitterbauer A, Kortuem B, Zimmermann M, Moser B, Bekos C, Steinlechner B, Hofbauer H, Klepetko W, Schenk P and Dome B

ABSTRACT

Background:
Although COPD is amongst the leading causes of morbidity and mortality worldwide, currently no biomarkers for early detection of COPD are known. We have recently demonstrated that manifest COPD is accompanied with elevated serum heat shock protein (HSP) 27 levels as compared to a control population.
Objectives:
We investigated whether elevated HSP27 levels are associated with early radiological signs of COPD (air trapping (AT) and emphysema (E)) and impaired lung function in an open prospective study.
Methods:
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 means of ELISA. Of those 120 subjects 94 underwent high resolution (HR) CT scan voluntarily.
Results:
The following results were attained in those 94 subjects undergoing HR-CT scan: AT or AT+E was detected in 57.45%. Subjects with AT+E (n=23) showed significantly higher HSP27 levels than those without any pathology (nothing abnormal detected, NAD) (4618±1677 vs. 3282±1607 pg/ml; mean±SD, P=0.0081). In a univariate logistic regression model including NAD and AT+E, the AUC of HSP27 in ROC curve was 0.724 (0.594-0.854 95% CI; P=0.0033). Interestingly, pro-inflammatory IL-8 was elevated in those subjects who evidenced AT+E as compared to AT and NAD. Lung function did not correlate with increased HSP27 levels and pathologic radiological findings.
Conclusions:
HSP27 serum levels positively correlated with early radiological signs of COPD, whereas lung function testing results did not match with radiological findings or HSP27 serum levels. Serum HSP27 level may serve as a potential marker to identify early signs of COPD independent of lung function in young smokers.
 

October 2011 Radio feature on OE1 on APOSEC

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"Hilfe zur Selbsthilfe bei Herzinfarkt. Forscher der Med-UniWien wollen körpereigene Reparaturmechanismen nutzen. Für Herzinfarkt-Akuttherapie werden weiße Blutkörperchen als Bioreaktoren für Medikamente genutzt."
Karin Pollack, Der Standard,  2011, Oct. 25/26. Article

Apoptotische Leukozyten und deren Sekretionsprodukt. Relevanz für die Herzinfarkttherapie beim Menschen.
Lichtenauer M, Mildner M, Gyöngyösi M, Ankersmit HJ. Wiener klinisches Magazin 5/2011. Article

"NEUER THERAPIEANSATZ: Keine bleibenden Schäden nach Herzinfarkt. In der Akutphase nach einem Herzinfarkt verabreichtes Proteinkonzentrat verringert entzündlich bedingte Vernarbung des Herzmuskels", Der Standard online, 2011, Oct. 6. Article

"Herzinfarkt: Erfolg mit neuer Therapie. Bleibende Schäden verhindern: Eine Erfindung Wiener Forscher könnte die Therapie von Herzinfarkt künftig entscheidend verbessern", Kurier online, 2011, Oct. 6. Article

September 2011 Paper accepted in Basic Research in Cardiology

Secretome of apoptotic peripheral blood cells (APOSEC) confers cytoprotection to cardiomyocytes and inhibits tissue remodelling after acute myocardial infarction: a preclinical study

Lichtenauer M*, Mildner M*, Hoetzenecker K, Zimmermann M, Podesser BK, Sipos W, Berényi E, Dworschak D, Tschachler E, Gyöngyösi M, Ankersmit HJ  *contributed equally to this work

Cell culture supernatants derived from irradiated apoptotic peripheral blood mononuclear cells (APOSEC) were collected and injected as a single dose intravenously after myocardial infarction in an experimental AMI rat model and in a porcine closed chest reperfused AMI model. Magnetic resonance imaging (MRI) and echocardiography were used to quantitate cardiac function. Analysis of soluble factors present in APOSEC was performed by enzyme-linked immunosorbent assay (ELISA) and activation of signalling cascades in human cardiomyocytes by APOSEC in vitro was studied by immunoblot analysis. 
Intravenous administration of a single dose of APOSEC resulted in a reduction of scar tissue formation in both AMI models. In the porcine reperfused AMI model APOSEC led to higher values of ejection fraction (57.0% vs. 40.5%, p<0.01), a better cardiac output (4.0 vs. 2.4 l/min., p<0.001) and a reduced extent of infarction size (12.6% vs. 6.9%, p<0.02) as determined by MRI. Exposure of primary human cardiac myocytes with APOSEC in vitro triggered the activation of pro-survival signalling-cascades (AKT, Erk1/2, CREB, c-Jun), increased anti-apoptotic gene products (Bcl-2, BAG1) and protected them from starvation induced cell death.
Intravenous infusion of culture supernatant of apoptotic PBMC attenuates myocardial remodelling in experimental AMI models. This effect is probably due to the activation of pro-survival signalling cascades in the affected cardiomyocytes. 

Figure Legend: Two representative images of porcine hearts explanted 30 days after AMI are shown. Hearts of APOSEC-injected animals evidenced only marginal formation of scar tissue in the myocardium compared to control animals where large infarcts were common.

May 2011 Recent publication in Basic Research in Cardiology was awarded the Österreichischen Kardiologenpreis (Basic Science, 1^st Place)

                                                Michael Lichtenauer received the award for best basic science publication in 2010/2011 from Prof. Irene Lang and DI Thomas Nowotny (BIOTRONIK) at the annual meeting of the Austrian Society of Cardiology (ÖKG) in Salzburg.

March 2011 Radio feature on OE1 on the new aspects of stem cell research 

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March 2011 Paper accepted in Basic Research in Cardiology

Intravenous and intramyocardial injection of apoptotic white blood cell suspensions prevents ventricular remodelling by increasing elastin expression in cardiac scar tissue after myocardial infarction.

Lichtenauer M, Mildner M, Baumgartner A, Hasun M, Werba G, Beer L, Altmann P, Roth G, Gyöngyösi M, Podesser BK, Ankersmit HJ.

Abstract
Congestive heart failure developing after acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Clinical trials of cell-based therapy after AMI evidenced only a moderate benefit. We could show previously that suspensions of apoptotic peripheral blood mononuclear cells (PBMC) are able to reduce myocardial damage in a rat model of AMI. Here we experimentally examined the biochemical mechanisms involved in preventing ventricular remodelling and preserving cardiac function after AMI. Cell suspensions of apoptotic cells were injected intravenously or intramyocardially after experimental AMI induced by coronary artery ligation in rats. Administration of cell culture medium or viable PBMC served as controls. Immunohistological analysis was performed to analyse the cellular infiltrate in the ischaemic myocardium. Cardiac function was quantified by echocardiography. Planimetry of the infarcted hearts showed a significant reduction of infarction size and an improvement of post AMI remodelling in rats treated with suspensions of apoptotic PBMC (injected either intravenously or intramoycardially). Moreover, these hearts evidenced enhanced homing of macrophages and cells staining positive for c-kit, FLK-1, IGF-I and FGF-2 as compared to controls. A major finding in this study further was that the ratio of elastic and collagenous fibres within the scar tissue was altered in a favourable fashion in rats injected with apoptotic cells. Intravenous or intramyocardial injection of apoptotic cell suspensions results in attenuation of myocardial remodelling after experimental AMI, preserves left ventricular function, increases homing of regenerative cells and alters the composition of cardiac scar tissue. The higher expression of elastic fibres provides passive energy to the cardiac scar tissue and results in prevention of ventricular remodelling.

Figure Legend: Hearts of apoptotic cell injected animals explanted six weeks after LAD ligation evidence less myocardial damage compared to controls. Hearts from medium or viable cell injected animals appear more dilated and show a greater extension of fibrotic tissue.

3 Abstracts accepted for ISHLT 2011 Congress in San Diego

Secretome of apoptotic peripheral blood cells (APOSEC) confers cytoprotection to cardiomyocytes and inhibits tissue remodelling after acute myocardial infarction

M. Lichtenauer, M. Mildner, K. Hoetzenecker, S. Hacker, M. Zimmermann, B. K. Podesser, W. Sipos, E. Berényi, E.Tschachler, M. Gyöngyösi, W. Klepetko, H. J. Ankersmit

Introduction: Heart failure following acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Our previous observation that injection of apoptotic peripheral blood mononuclear cells (PBMC) was able to restore long-term cardiac function in a rat acute ischemia model prompted us to study the effect of soluble factors derived from apoptotic PBMC on ventricular remodelling after AMI.
Methods: Cell culture supernatants derived from irradiated apoptotic peripheral blood mononuclear cells (APOSEC) were collected and injected as a single dose intravenously after myocardial infarction in an experimental AMI rat model and in a porcine closed chest reperfused AMI model. Magnetic resonance imaging and echocardiography were used to quantitate cardiac function. Immunohistology and flow cytometry were used to analyse the cellular components of the affected cardiac sites. Analysis of soluble factors present in APOSEC was performed with membrane arrays and activation of signalling cascades in human cardiomyocytes by APOSEC in vitro was studied by immunoblot analysis. 
Results: Intravenous administration of a single dose of APOSEC resulted in a reduction of scar tissue formation in both AMI models. In the porcine reperfused AMI model APOSEC led to higher values of ejection fraction (57.0% vs. 40.5%, p<0.01), a better cardiac output (4.0 vs. 2.4 l/min., p<0.001) and a reduced extent of infarction size (12.6% vs. 6.9%, p<0.02) as determined by MRI. Administration of APOSEC in the rat AMI model caused increased presence of CD68+ macrophages and c-kit+ endothelial progenitor cells (EPC) in the infarcted myocardium within 72 hours. Exposure of primary human cardiac myocytes with APOSEC in vitro triggered the activation of pro-survival signalling-cascades (AKT, p38 MAPK, Erk1/2, CREB, c-Jun) and increased anti-apoptotic gene products (Bcl-2, BAG1).
Conclusions: Intravenous infusion of culture supernatant of apoptotic PBMC attenuated myocardial remodelling in both models of experimental AMI. This effect seems to be due to the activation of pro-survival signalling cascades in the affected cardiomyocytes and to a higher presence of regenerative cells (EPC and macrophages) within the ischemic tissue. 

Administration of anti-thymocyte globulin (ATG) preserves cardiac function after experimental myocardial infarction

M. Lichtenauer, G. Werba, M. Mildner, M. Hasun, A. Baumgartner, S. Nickl, M. Rauch, M. Zimmermann, B. K. Podesser, W. Klepetko, H. J. Ankersmit

Introduction: Despite advances in clinical interventions and drug therapy for cardiovascular disease, ventricular remodeling after myocardial infarction (MI) and congestive heart failure continues to be a significant medical problem. Over the last decades research has focused on finding therapies to reduce inflammatory reactions after an ischemic event. Of relevance are reports showing that infusion of apoptotic leucocytes or anti-lymphocyte serum after MI can reduce myocardial necrosis and preserves cardiac function. In order to corroborate this therapeutic mechanism, the utilisation of immunosuppressive agents with a comparable mechanism such as anti-thymocyte globulin (ATG) was evaluated in this study.
Methods: For in vivo experiments, MI was induced in rats by ligation of the left anterior descending artery. Initially after the onset of ischemia, rabbit ATG (10mg/rat) was injected intravenously. Untreated and sham operated animals served as controls. Histological evaluations were performed three days after MI in order to analyze angiogenic cell populations in the infarcted myocardium. Cardiac function was analyzed by echocardiography six weeks after induction of MI. Determination of infarction size was conducted by planimetry. 
Results: Rats that were injected with ATG evidenced higher numbers of CD68+ macrophages and c-kit+ endothelial progenitor cells (EPC) in the ischemic myocardium 72 hours after MI as compared to controls. Animals injected with ATG evidenced less myocardial necrosis, showed a significant reduction of infarct dimension and an improvement of post MI remodeling after six weeks (infarct dimension 26.5% vs. 12.5%, p<0.01). Furthermore, echocardiography revealed an improved functional recovery in treated animals as evidenced by a reduced loss of ejection fraction (EF, 43% in controls vs. 53% in treated animals, p<0.01, n=13 per group).
Conclusions: These data indicate that ATG, a therapeutic agent successfully applied in clinical transplant immunology, salvaged ischemic myocardium, increased the homing of macrophages and EPC and improved cardiac function after experimental MI in rats.

Intramyocardial injection of irradiated apoptotic peripheral blood mononuclear cells (PBMC) preserves ventricular function after myocardial infarction

M. Lichtenauer, K. Hoetzenecker, M. Hasun, A. Baumgartner, M. Mildner, S. Nickl, G. Werba, M. Zimmermann, A. Mitterbauer, B. K. Podesser, W. Klepetko, H. J. Ankersmit

Background: Acute myocardial infarction (AMI) followed by cardiac remodeling is a major cause of congestive heart failure and death. Of clinical relevance are reports that demonstrated that infusion of apoptotic cells lead to an initiation of immunosuppressive mechanisms. Based on these reports, we hypothesized that injection of apoptotic cells into ischemic myocardium reduces inflammatory reactions after AMI. 
Methods: Cell suspensions of irradiated apoptotic PBMC were injected intramyocardially (IM) in an experimental rat model of AMI. Sham operated animals and rats injected with control medium or viable cells served as controls. Tissue specimens were obtained 72 hours after induction of AMI to analyse the cellular infiltrate within the ischemic myocardium. Cardiac function was analysed by echocardiography and infarction size was determined by planimetry after six weeks. The composition of the fibrotic scar was analysed for elastic and collagenous fibers by using the Elastica van Gieson staining.
Results: Rats that were injected with irradiated apoptotic PBMC showed enhanced homing of macrophages and endothelial progenitor cells (EPC) within 72 hours as compared to controls. A histological evaluation showed a higher ratio of elastic fibers in the infarct border zone of animals injected with apoptotic cells (p<0.01). Planimetric analysis showed a significant reduction of infarction size and improvement of post AMI remodeling with less signs of dilation (infarct dimension 9.1% in IM injected rats, 24.9% in controls, p<0.001, respectively). Echocardiography revealed that ventricular function was almost preserved in the treatment group with EF values of 55% in treated animals vs. 42% in untreated controls compared to 61% in sham operated rats (n=13 per group, p<0.01).
Conclusions: These data indicate that irradiated apoptotic PBMC suspensions administered IM caused the homing of regenerative EPC, led to higher numbers of macrophages 72 hours after AMI, altered the elastin/collagen ratio within the scar in a positive manner and preserved cardiac function.

Interview on "How to modulate inflammatory conditions" published in the daily newspaper "Der Standard" (Nov. 30, 2010)

-> Link "Interview: Entzündungsprozesse modulieren"

February 2011 Paper published in American Laboratory

Effect of PBS Solutions on Chemokine Secretion of Human Peripheral Blood Mononuclear Cells

Lichtenauer M, Nickl S, Hoetzenecker K, Mangold A, Mitterbauer A, Hacker S, Zimmermann M, Ankersmit HJ.

Phosphate buffered saline (PBS) solutions are commonly used in laboratories for dilutions, washing cell suspensions, rinsing as well as an additive to cell culture media. In the present study we evaluated chemokine secretion of peripheral blood mononuclear cells (PBMC) incubated in medium containing PBS solutions with varied concentrations of calcium and magnesium. A dose dependent increase of Interleukin-8 (IL-8) was found when PBMC were exposed to increasing concentrations of +/+ PBS. As extracellular calcium gradients have been attributed to induce cellular migration, the secretion of chemokines suggests an important additional modulatory mechanism of heightened calcium levels in cell culture conditions. 

The figure above shows a dose dependent effect of supplementing PBMC cell cultures with increasing concentrations of calcium and magnesium containing PBS, resulting in heightened levels of IL-8. * p<0.05 vs. control

May 2010 Research Update accepted in the European Journal of Clinical Investigation

Research Update: Myocardial lipofuscin-laden lysosomes contain the apoptosis marker caspase-cleaved cytokeratin-18

Lichtenauer M and Ankersmit HJ

In our previous work we provided evidence that myocardial lipofuscin is bearing caspase-cleaved cytokeratin 18 (ccCK-18), a marker for apoptotic cell death, predominantly of epithelial derived cells. This finding closely correlated with myocardial pathologies as ischemic, hypertrophic and congestive cardiomyopathy. As a feature of apoptotic cell turnover in the myocardium during myocardial infarction we also detected elevated systemic levels of ccCK-18 in patients’ serum after myocardial ischemia. As these disease entities are more prevalent in patients suffering from chronic obstructive pulmonary disease (COPD), we investigated the issue of ccCK-18 and its correlation with disease severity in COPD. We were able to show elevated serum levels ccCK-18 among patients with COPD GOLD stage I&II (295 units/l p>0.008) and COPD III&IV (465 units/l p>0.001) compared to smokers without COPD (120 units/l) substantiating an apoptosis-dependent lung degeneration leading to a decline of lung function and finally emphysema. In line with our previous data, this indicates the significance of ccCK-18 as a convenient biomarker for the diagnosis of both cardiac and pulmonary pathologies.

Congress Announcement

2nd EACTS Meeting on Cardiac and Pulmonary Regeneration


Courtyard Vienna Messe Hotel, Vienna, Austria
3-4 December 2010

On behalf of the local organizing committee we would like to propose the 2nd EACTS Cardiac and Pulmonary Regeneration meeting, in Vienna, Austria. After the overwhelming success of the 1st meeting in 2008 in Bern, Switzerland; once again, this meeting will be dedicated to the emerging field of regenerative medicine for cardiac and pulmonary repair and regeneration. 

We propose a two-day symposium with state-of-the-art presentations by invited experts in regenerative medicine and aim to cultivate a friendly atmosphere with ample time for discussion and presentations. This meeting will be very interactive with the goal not only of being educational but also of fostering collaborations. We have selected a great panel of experts from the cardiovascular and pulmonary regeneration fields to discuss topics such as: 

Stem Cell Types for Therapeutic Strategies 
Importance of Microenvironment & Inflammation in Heart & Lung 
Cell-Based Therapy for Cardiovascular & Pulmonary Regeneration 
Growth Factor-/Secretome-Based Therapeutics for Heart & Lung Regeneration 
Regeneration in the Skin – Lessons for the Heart and Lung


Registration - coming soon


Scientific Committee
Prof. Thomas Geiser, (University Hospital Berne),
Dr. Marie-Nöelle Giraud, (University Hospital Berne),
Prof. Mariann Gyöngyösi, (Med. Univ. Vienna)
Prof. Irene Lang, (Med. Univ. Vienna)
Prof. Günther Laufer, (Med. Univ. Vienna)
Prof. Gerald Maurer, (Med. Univ. Vienna)
Prof. Hendrik Tevaearai, (University Hospital Berne),
Prof. Erwin Tschachler, (Med. Univ. Vienna)
Prof. Rolf Ziesche, (Med. Univ. Vienna)

Chairs
Dr. Hendrik J. Ankersmit, (Med. Univ. Vienna)
Dr. Golnaz Karoubi, (University Hospital Berne)

May 2010 Paper accepted in Interactive Cardiovascular & Thoracic Surgery

CASE REPORT: Spontaneous rupture of the inferior thyroid artery resulting in mediastinal hematoma

Hoetzenecker K, Töpker M, Klepetko W, Ankersmit HJ

In obstructive urinary tract disorders, the Valsalva maneuver can be performed in order to ease micturition. Among other things, the maneuver leads to complex vascular reactions and increases the systemic blood pressure. These vascular changes pose a serious stressor to the vessel wall and ruptures of small arteries have been anecdotically described in literature. Herein, we present a case of a patient referred to a thoracic surgeon with a huge mediastinal hematoma. Diagnostic work-up revealed a spontaneous rupture of the left inferior thyroid artery due to repetitive Valsalva maneuver.

April 2010 Paper accepted in the Transplant International

CASE REPORT: Considerations on infectious complications using a drowned lung for transplantation

Hoetzenecker K, Ankersmit HJ*, Lang G, Scheed A, Marta G, Jaksch P, Klepetko W

*Corresponding author

Summary: Recently, the applicability of lungs from drowned victims for transplantation has been anecdotically described in literature. However, no data exist about hazards or limitations. Herein, we describe a case of lung transplantation from a submersion victim and the subsequent development of an Aeromonas hydrophila infection in the implanted organ. Based on this case we propose standard procedures, which should be followed when considering drowned donor lungs, in order to minimize risks for infectious complications.

March 2010 Paper accepted in the Journal of Thoracic Oncology

Apelin expression in human non-small cell lung cancer: Role in angiogenesis and prognosis

Berta J, Kenessey I, Dobos J, Tovari J, Klepetko W, Ankersmit HJ, Hegedus B, Renyi-Vamos F, Varga J, Lorincz Z, Paku S, Ostoros G, Rozsas A, Timar J, Dome B.

Introduction: The recently discovered bioactive peptide, apelin, has been demonstrated to stimulate angiogenesis in various experimental systems. However, its clinical significance and role in tumor vascularization have not yet been investigated in a human malignancy. Therefore, our aim was to study whether apelin expression is associated with angiogenesis and/or tumor growth/behavior in human non-small cell lung cancer (NSCLC).
Methods: 94 patients with stage I-IIIA NSCLC and complete follow-up information were included. Apelin expression in human NSCLC samples and cell lines was measured by quantitative RT-PCR, ELISA and immunohistochemistry. Effects of exogenous apelin and apelin transfection were studied on NSCLC cell lines in vitro. In vivo growth of tumors expressing apelin or control vectors were also assessed. Morphometric variables of human and mouse tumor capillaries were determined by anti-CD31 labeling. 
Results: Apelin was expressed in all of the six investigated NSCLC cell lines both at the mRNA and protein levels. Although apelin overexpression or apelin treatments did not increase NSCLC cell proliferation in vitro, increasing apelin levels by gene transfer to NSCLC cells significantly stimulated tumor growth and microvessel densities and perimeters in vivo. Apelin mRNA levels were significantly increased in human NSCLC samples when compared to normal lung tissue, and high apelin protein levels were associated with elevated microvessel densities and poor overall survival.
Conclusions: The current study reveals apelin as a novel angiogenic factor in human NSCLC. Moreover, it also provides the first evidence for a direct association of apelin expression with clinical outcome in a human cancer.

March 2010 Paper accepted in the Journal of Surgical Research

Upregulation of Interleukin 33 and soluble ST2 serum levels in liver failure

Roth G*, Zimmermann M, Lubsczyk B, Pilz J, Faybik P, Hetz H, Hacker S, Mangold A, Bacher A, Krenn CG, Ankersmit HJ

* Corresponding Author

Background: IL-33, a member of the IL-1 family induces the production of proinflammatory and Th2-associated cytokines and may also serve as an 'alarmin' similar to HMGB1. Soluble ST2 has been implicated as a decoy receptor, to attenuate Th2 inflammatory responses. The relevance of both molecules in hepatic failure is unknown. 
Materials and Methods: The trial was a prospective preliminary study in a university hospital surgical ICU. Eleven patients with acute liver failure (ALF) and twelve patients with acute-on-chronic liver failure (ACLF), who were admitted to the ICU. 14 patients with chronic hepatic failure (CHF) awaiting liver transplantation and 13 healthy individuals served as controls. IL-33 and soluble ST2 concentrations were determined by ELISA. 
Results: The concentration of IL-33 and soluble ST2 was significantly higher in ALF, ACLF and CHF patients as compared to the controls. Soluble ST2 serum concentration was significantly elevated in ALF and ACLF as compared CHF, moreover soluble ST2 was significantly higher in CHF as compared to healthy controls. IL-33 and soluble ST2 serum levels correlated significantly (r = 0.6117, P < 0.0001). Moreover there was a correlation between IL-33 serum levels and alanine aminotransferase (ALT) activity in CHF, ALF and ACLF patients (r = 0.4321, P = 0.0171).
Conclusion: Our data provide evidence for elevated levels of IL-33 and soluble ST2 in liver failure, which could a sign of immune hyperactivation, and / or a mechanism to downregulate inflammation. Especially, soluble ST2 maybe useful to discern acute from chronic hepatic failure or to monitor the course and the severity of the disease.

Panel A and B: Concentration of serum IL-33 and soluble ST2 in healthy controls (n = 13), chronic hepatic failure (CHF, n = 14), acute liver failure (ALF, n = 11) and acute-on-chronic liver failure (ACLF, n = 12,). Each box represents the inter quartile containing the 50% values. The line across the box indicates the median line. The whiskers extend from the box to the 5th and 95th percent.

March 2010 Paper accepted in Cardiovascular Research

Primary sources and immunological prerequisites for sST2 secretion in humans

Mildner M, Storka A, Lichtenauer M, Mlitz V, Ghannadan M, Hoetzenecker K, Nickl S, Dome B, Tschachler E, Ankersmit HJ

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-33 (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. Strongest sST2 mRNA expression was detected in heart and lung tissues, which correlated with spontaneous secretion of sST2 protein in vitro. The inflammatory cytokines interleukin-1a (IL-1a), interleukin-1b (IL-1b) and tumor necrosis factor a (TNF a) as well as supernatants of lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMC) led to an enhanced secretion of sST2 in cultured cardiac myocytes and lung alveolar epithelial cells. These cytokines enhanced sST2 secretion via a NFkB-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.

Scheme of the human endotoxin model. Endotoxin stimulation leads to inflammation and the production of inflammatory cytokines such as IL-1a, IL-1b, IL-6 and TNFa. Inflammatory cytokines stimulate both lung alveolar epithelial cells and cardiac myocytes to an enhanced sST2 production via a NFkB dependent mechanism. Secreted sST2 enters the circulation and consequently contributes to the attenuation of immune responses in organs exposed to environmental and autologous antigenic triggers.

March 2010 Letter published in the Journal of The International Society for Heart and Lung Transplantation (ISHLT)

Mangold A, Ankersmit HJ

Comment on: Lila N, McGregor CG, Carpentier S, Rancic J, Byrne GW, Carpentier A. 
Gal knockout pig pericardium: New source of material for heart valve bioprostheses. J Heart Lung Transplant. 2009 ahead of print 

In the study presented recently by Lila et al., they evaluated the calcification tendency of alpha-Gal-containing porcine pericardium tissue compared to pericardium from alpha-Gal-knockout (KO) pigs. As our data provided the basis for that work, we would like to comment on it. In 2005, we described alpha-Gal epitopes in glutaraldehyde (GA)-fixed bioprosthetic heart valves for the first time and denitifely disproved the often raised assertion that the fixation process of biological valves is abolishing antigenicity of the tissue. Furthermore, we were able to detect a significant elevation of anti-alpha-Gal IgM antibodies in bioprostheses recipients 10 days after operation. Recently, we showed that alpha-Gal-specific IgG antibodies are significantly increased in patients' sera three months after bioprosthetic valve implantation. This is the proof for a long-lasting immune response to alpha-Gal residues within the valve tissue. In biovalves explanted after remaining one year in circulation, we neither detected alpha-Gal-positive structures nor any cell nuclei, indicating that these cells had been eectively degraded while being exposed to the blood circuit. In detail, Lila et al. implanted GA-fixed tissue, GA-fixed + formaldehyde, ethanol and Tween 80 (FET)-treated tissue, with and without preincubation with human anti-Gal antibodies subcutaneously into Wistar rats during one month. They found a significant reduction of calcification of Gal-KO pericardium compared to wild-type tissue. Further, calcification was greatly reduced through addition of FET. When the implants were pre-incubated with human anti-Gal antibodies, Gal-positive tissue revealed to have strongly enhanced calcification compared to Gal-negative tissue, or any tissue not pre-incubated with such antibodies. These interesting results need to be discussed in more detail. What puzzled us is the methologic concept chosen in this work: (a) The tissue samples were implanted subcutaneously. The authors explain that this method is adequate as it is traditionally used and empirically well validated. This argument is not convincing, as the environment is physiologically simply different. Manji et al. delivered distinct data for xenograft calcification by implantation in rats into the infrarenal position, compared to former subcutaneous models. (b) As correctly reviewed by Lila, humans and old world monkeys are the only mammals who lack the alpha-Gal epitope but display high anti-Gal titers. Thus, rats are the wrong model, if the immunological role of alpha-Gal wants to be addressed. Rats express the Gal epitope themselves, and do not have anti-Gal antibodies. Therefore, a specific immune response to alpha-Gal is hardly possible. (c) The preincubation with human anti-Gal antibodies is an approach towards an immunological linkage; but the implicated assumption of the authors, that antibodies can accomplish effector functions in a xenogenic system such as human antibodies in a rat organism is unsustainable. This may be possible, but the actual scenario taking place can never be defined clearly, and hasn't even been tried in the present study. In so far, the presented results are lacking explanation. Nevertheless, they are at hand; it would be restrictive not to consider them. We know that alpha-Gal epitopes and the respective antibodies can differ slightly amongst each other, this maybe could be a possible explanation favoring an immunological background. However, this is unlikely, from our point of view, and would have to be proven. As immunological reasons for the signifcant difference in calcification of alpha-Gal-negative tissue must be questioned, other possible explanations should be evaluated. We are contented that our results are encouraging researchers to seek for deeper insights concerning alpha-Gal and its role in bioprosthetic heart valve destruction, but emerging obstacles shall be overcome rather than evaded.

January 2010  Reply Letter published in the Journal of Thoracic and Cardiovascular Surgery

What is the Real Risk of Stent-Graft Infection in the Treatment of Aortobronchial Fistulas?

Ankersmit HJ

Reply to the Editor:
Bozzani and colleagues state that surgical correction of an aortobronchial fistula, particularly open correction of a thoracic aneurysm, carries a fairly high postoperative incidence of stent-graft infection. To the contrary, minimal infection rates were observed after endovascular stent placement. The authors question whether antibiotic therapy should be administrated after this minimally invasive operational procedure.There is scarce literature on immunologic consequences after stent implantation in humans. According to immunologic data obtained from patients undergoing heart operations with cardiopulmonary support and abdominal surgery, any operation performed in humans induces a state of immune suppression in vivo. Therefore, patients undergoing heart surgery (cardiopulmonary support) should receive aggressive 5-day antibiotic treatment in accordance with the insight of an induced "systemic immune suppression" after heart surgery. In regard to the ongoing discussion of antibiotic treatment after endovascular stent implantation, the following approach seems to be feasible. Studies have to be initiated to investigate the immunologic consequence of open and endovascular stent implantation in humans (eg, abdominal aorta aneurysm repair, open, closed), and ‘‘yes,’’ antibiotic treatment should be applied for 4 to 5 days after endovascular stent placement to potentially "prohibit" pain from infection (local, systemic) in patients.

September 2009  Reply Letter published in the Journal of Thoracic and Cardiovascular Surgery

Pulmonary Arterial Hypertension and Congenital Heart Disease: Targeted Therapies and Operability

Hoetzenecker K, Ankersmit HJ, Lang IM

Reply to the Editor:
We thank Dr Beghetti and colleagues for their thoughtful comments regarding the hemodynamic evaluation and further management of our recent case of atrial septal defect (ASD). The management of patients with a degree of pulmonary vascular disease prior to shunt closure has been a matter of debate. In addition, recent studies demonstrating the efficacy of oral vasodilators in pulmonary vascular disease associated with congenital systemic-to-pulmonary shunts have fueled an uncertainty of vasodilator pretreatments prior to shunt closure. Despite optimized medical treatment with diuretics, antibiotics, and oral anticoagulant over 4 months since the first medical contact, our patient was severely dyspneic, with elevated atrial pressures, a pro–brain natriuretic peptide serum level of above 4000 pg/mL, and a dramatically limited 6-minute walking distance (6-MWD) of 150 m. In fact, based on the hemodynamic assessment alone, the patient was admitted to the surgical ward for ASD closure. However, surgeons refused the operation based on the patient’s overall clinical profile and frailty. A 10-month treatment with bosentan on top of supportive treatment with diuretics and anticoagulation effectively decreased shunt flow and lowered pulmonary vascular resistance by 140 dynes . s-1 . cm-5 and markedly decreased atrial pressures, biomarkers, and 6-MWD in the presence of a mild arterial desaturation. We do agree with the discussants that taking into account left atrial pressures, pulmonary arteriolar resistance was about 3 Woods. In addition, the pulmonary-to-systemic resistance ratio under oxygen and nitric oxide was <0.33 (in the patient, this ratio was 0.11), a threshold pediatric cardiologists have labeled as a criterion conveying a good prognosis after closure of the shunt. Still, data in adult patients with congenital heart disease are lacking, and the criteria of a complete hemodynamic responder status in adults were not fulfilled in this case. Because hemodynamic testing is a routine procedure in adult pulmonary vascular centers, we do rely on these data in the absence of firm evidence indicating their uselessness in adults with congenital heart disease. Moreover, children are usually examined under general sedation/anesthesia. For these and other reasons, it is evident that the hemodynamic response pattern in children is different from that in adults6 and that hemodynamic criteria in children may not apply to elderly adults. Furthermore, later assessments after surgery in the patient under discussion illustrated a degree of persistent pulmonary vascular disease with a pulmonary arteriolar resistance of 530 dynes . s-1 . cm-5, despite active treatment with bosentan. The main value of this report is to provoke discussion, because due to its single case nature, surgery in the absence of bosentan cannot be repeated. We submit that our invasive procedure Was based on an integrative clinical and hemodynamic approach and guided by numbers, rather than the reverse. Controlled data to guide a "targeted treatment-and-repair" strategy in adult patients with congenital heart disease are needed.

The table shows the percentage of CD4+CD28null cells in the peripheral blood flow. Furthermore, cytokine expression in supernatants of PBMCs stimulated with either anti-CD3 or PHA is described. All date are given as mean (95% confidence interval).

July 2008  Paper accepted in the European Journal of Clinical Investigation

Background: Intravenous immunoglobulins (IVIg) and Cytomegalovirus immunoglobulins (CMVIg) are currently finding increased acceptance in clinical states of high immune activity and in transplant recipients. A rare side effect of their application is intravascular thrombosis, which is thought to be related to pre-existing hyperviscosity. In a previous study we have shown that rabbit antithymocyte globuline (rATG) causes platelet aggregation in vitro via the Fc IgG receptor (CD32). 
Objectives: To investigate if IVIg and CMVIg have the potential to cause CD32 dependent platelet aggregation.
Methods: The influence of CMVIg or IVIg on platelets pre-incubated with or without monoclonal antibody AT10 was studied in an aggregometer. Expression of platelet surface activation marker CD62P was determined by FACS analysis and presence of soluble CD40L (sCD40L) was evaluated by ELISA. All in vitro experiments were performed using platelet concentrates from the blood bank, at therapeutic concentrations of immunoglobulins.
Results: Incubation of platelets with CMVIg and IVIg markedly induced platelet aggregation, increased expression of CD62P and secretion of sCD40L. The capacity of CMVIg and IVIg to induce platelet aggregation was completely abrogated by adding the blocking antibody AT10 directed against the low-affinity Fc IgG receptor (CD32).
Conclusions: Our results suggest that CMVIg and IVIg solutions with activating Fc domains are able to bind CD32 on platelets and cause platelet aggregation in vitro. These results indicate a mechanism by which in vivo intravasal thrombosis may be explained and suggest caution with concomitant use of packed platelets and IVIg in autoimmune diseases in the clinical setting.

January 2008  Case Report accepted in The Middle European Journal of Medicine (Wiener Klinische Wochenschrift)

Gigantic Coronary Fistula 

A 43-year-old male patient without clinical symptoms presented an enlarged heart shadow in a routine radiological examination. Computed tomography (CT) revealed a structure in the pericardial sac that was initially classified as a pericardial cyst. In order to confirm the diagnosis, an electrocardiogram (ECG) -triggered multi-slice CT was performed resulting in the diagnosis of a gigantic coronary fistula originating from the left main coronary artery leading to the right atrium (Figure 1; solid arrows: Fistula; asterisk: Descending Thoracic Aorta). The shunt volume of the coronary fistula was estimated to be 50%. Echocardiography demonstrated dilatation of the right chambers due to volume overload. 
Since operative mortality was deemed extremely low in this patient surgical correction was advised. After median thoracotomy, initiation of heart lung machine and extensive cardioplegia, the coronary fistula was found to originate from the left main coronary artery meandering around the posterior side of the left heart with a mean diameter of 2 cm and entering the right atrium at the level of the vena cava superior (Figure 2; solid arrows: Fistula). The fistula was ligated in the right atrium and at its origin at the branching site of the circumflex artery. To secure optimal surgical outcome bypass grafting was performed to left anterior descending (LAD) and its diagonal branch as well as the circumflex artery. Postoperatively performed ECG-triggered multislice-CT evidenced successful repair of this anatomical malformation. The postoperative course was uneventful.

September 2007 Paper accepted in the European Journal of Clinical Investigation

Cytomegalovirus hyperimmunoglobulin: Mechanisms in Allo-Immune Response in vitro

Background: Cytomegalovirus hyperimmunoglobulin (CMVIg) containing drugs are routinely administered in cardiac transplantation for prophylaxis against CMV disease. Yet little is known about their influence on transplant relevant immune functions. The aim of this study was to evaluate the effect of CMVIg on cellular immunity in in vitro experiments and to define their role in tolerance inducing mechanisms. 
Materials and Methods/Results: CMVIg reduces proliferation in mixed lymphocyte reactions and anti-CD3 blastogenesis assays and is related to decreased production of immune modulating cytokines interleukin (IL)-2, interferon (IFN) γ, IL-10. This anti-proliferative effect is associated with a cell-cycle arrest in the G0/G1 phase and induction of apoptosis in CD8+ and natural killer cells. Co-incubation with CMVIg causes downregulation of cell bound immunoglobulin and FcγRIII surface expression on natural killer cells and leads to attenuation of antibody dependent cellular cytotoxicity effector functions. 
Conclusions: We conclude that CMVIg induces immunological features on leukocytes in vitro that are known to be related to tolerance induction. Our observations extend the current concept of CMVIg as passive CMV prophylaxis to a therapeutic drug compound capable to reduce allogeneic immune response.

July 2007 Paper accepted in Annals of Thoracic Surgery

Heat Shock Proteins 27, 60, 70, 90α and 20S Proteasome in  On- versus Off-Pump Coronary Artery Bypass Graft Patients 

Background: The secretion of heat shock protein (HSP) 27, HSP60, HSP70, HSP90α, 20S proteasome, and their correlations to proinflammatory cytokine interleukin-6 is unknown in patients undergoing on-pump versus offpump coronary artery bypass graft (CABG) operation. Methods. Forty patients were included in this explorative study (on- versus off-pump CABG, each n=20). Serum samples were obtained before and 30 minutes, 60 minutes, and 24 hours after CABG operation. Enzymelinked immunosorbent assay technique was utilized to determine soluble HSP27, 60, 70, and 90α, 20S proteasome, and levels of interleukin-6. Results. Serum levels of HSP are increased in patients undergoing on-pump CABG operation as compared with off-pump CABG technique. These differences were highly significant for HSP27, 70, and 90α, at 60 minutes after initiation of cardiopulmonary bypass (all, p < 0.001). Concentrations of soluble 20S proteasome were increased 24 hours after operation in on- and off-pump CABG patients (p < 0.001) and correlated significantly with the serum content of HSP 27, 70, and 90α at 60 minutes after initiation of cardiopulmonary bypass (p < 0.001). No correlation was found when comparing interleukin-6 levels with intravascular leakage of HSP and 20S proteasome after CABG operation. Conclusions. We conclude from our data that the innate immune system is activated owing to spillage of known immune modulatory and apoptosis-associated proteins after CABG operation.

May 2007  Paper accepted in the Journal of Clinical Laboratory Analysis

Caspase-Cleaved Cytokeratin 18 and 20S Proteasome in Liver Degeneration

Apoptosis of epithelial hepatocytes plays a pivotal role in acute as well as in chronic liver diseases. The cleavage of cytokeratin-18 (CK-18) by caspases is an early event in the apoptotic process. We therefore sought to investigate serum levels of CK-18 and 20S proteasome in patients with liver cirrhosis progressive graft dysfunction, and acute liver failure (ALF), and in healthy volunteers. Enzyme-linked immosorbent assay (ELISA) was utilized to measure the concentration of M30, a fragment of CK-18 cleaved at Asp396 (M30 neoantigen), and the concentration of 20S proteasome, Serum levels of the CK18 neoepitope M30 were significantly increased in ALF, primary graft dysfunction (PDF), and liver cirrhosis vs. healthy controls (1,993.6±124.7 U/L, 2,238.1±235.9 U/L, and 673.6±86.5 U/L vs. 66.8±29.1 U/L, respectively P<0.001). Similar results were detected with the evaluation of 20S proteasome (124,014.5±13,235.6 ng/mL, respectively; P<0.001). Detection of CK-18 neoepitope M30 and 20S proteasome may represent a novel marker of tracing apoptotic epithelium, respectively mirroring degenerative liver processes in affected patient population. 

February 2007 Paper accepted in the European Journal of Clinical Investigation                                     

Elevated levels of interleukin-1beta-converting enzyme and caspase-cleaved cytokeratin-18 in acute myocardial infarction 

Systemic inflammation and apoptosis-specific immune activation play a major role in acute coronary syndromes (ACS) including acute myocardial infarction (AMI). The role of systemic and coronary obtained inflammatory plasma protein interleukin-1beta precursor (IL-1betap), IL-1beta-converting enzyme (ICE) and the apoptosis-specific caspase-cleaved cytokeratin-18 (ccCK-18) are not known in ACS. Plasma samples were obtained from stable angina, unstable angina and patients with AMI. Coronary blood was acquired by means of thrombectomy devices (X-sizer) in AMI patients. IL-1betap, ICE and ccCK-18 were determined by enzyme-linked immunosorbent assay. Multivariate logistic regression analysis was performed to determine predictive values of IL-1betap, ICE and ccCK-18 as compared to creatine kinase (CK) and troponin T (TnT) in order to relate these markers with the occurrence of myocardial damage. Our data suggest that ACS is related to increased concentration of systemic soluble ICE and ccCK-18. Moreover, soluble ccCK-18 was identified to be a superior marker as compared to TnT or CK, for detection of myocardial damage.

October 2006 Paper accepted in the American Journal of Transplantation

Blockade of RAGE suppresses alloimmune reactions in vitro and delays allograft rejection in murine heart transplantation.  

The receptor for advanced glycation endproducts (RAGE), a multiligand member of the immunoglobulin superfamily, interacts with proinflammatory AGEs, the products of nonenzymatic glycation and oxidation of proteins; high-mobility group box 1 (HMGB1), also known as amphoterin and S100/calgranulins to amplify inflammation and tissue injury. Previous studies showed that blockade of RAGE suppressed recruitment of proinflammatory mechanisms in murine models. We tested the hypothesis that RAGE contributes to alloimmune responses and report that in vivo, acute rejection of fully allogeneic cardiac allografts in a murine model of heterotopic cardiac transplantation is significantly delayed by pharmacological antagonism of RAGE. In parallel, allogeneic T-cell proliferation in the mixed lymphocyte reaction is, at least in part, RAGE-dependent. These data provide the first insights into key roles for RAGE in allorecognition responses and suggest that antagonism of this receptor may exert beneficial effects in allogeneic organ transplantation.