Sepsis-associated Microthrombus Formation: Processing and Secretion of von-Willebrand Factor Cleaving Protease

Acronym: ADAMTS13

Principal Investigator: Michael Ekaney 

Scientific Advisor: PD Dr. RA Claus

Research Area: D Sepsis related Organ Failure

Project Number: D1.9

Duration: 01.05.2011 - 30.04.2014

Module: PhD Fellowship

The Problem

Reduced ADAMTS13 is a hallmark of sepsis-associated coagulopathy with increased ULVWF-platelet aggregation and deposition of microthrombi. In this project, we investigate events of endothelial dysfunction affecting ADAMTS13 synthesis and activity in septic conditions.

Results so far

We hypothesized that extracellular histones released during sepsis are triggers of endothelial dysfunction. In a clinical setting, we have shown that histones are significantly elevated through out the course of sepsis and are found associated with thrombocytopenia and renal dysfunction. Patients who received APC treatment showed a decrease in histone levels. In a cell specific approach, we observed a dose dependent decrease in cell viability after histone stimulation and APC conciliated effects. LDH release was also dose dependently increased. Real-time Impedance response of HMEC-1 cells after stimulation with histones decreased and APC non-responsive to this effect. We also found that ADAMTS13 transcription markedly declined after 24h in the presence of proinflammatory cytokines, LPS and septic serum while septic patients who received APC showed an increase in ADAMTS13 activity.

Contact

Dr. Ralf Claus

Michael Ekaney

Tel. +49 (0)3641 - 9 32 58 60

Universitätsklinikum Jena
AG Molekulare Mechanismen des Organversagens
Forschungszentrum Lobeda
Am Klinikum 1
07747 Jena

Pathogen-Host Interaction during Infection with Candida albicans and its Progression to Sepsis

Acronym: Candida

Principal Investigators: Dr. med. Falk A. Gonnert, Prof. Bernhard Hube

Team: Dr. Sascha Brunke, Dr. Ralf Claus, Nayla Jbeily (MSc)

Research Area: D Sepsis related Organ Failure

Project Number: D1.5

Duration: 01.08.2010 - 31.07.2015

Module: Rotational Position, Start-up Scientist      

The Problem

Candida albicans dissemination via the bloodstream leads to systemic infections and sepsis. However, the early steps of attachment to and invasion into endothelia and subsequent organ colonization by C. albicans are largely unknown. This project aims to elucidate these early processes by using intravital microscopy.

Results so far

Detection of disseminating Candida albicans yeasts in live animals required the construction of effciently labelled fungal strains. Since standard GFP-labelled strains are not suitable in our system, we tagged a commonly used clinical isolate of C. albicans with the fluorescent mCherry protein for in vivo visualization. In vitro experiments showed that using the more effcient TDH3 promotor instead of the commonly used ACT1 system increased fluorescent by a factor of 10, which proved to be essential for the image acquisition in vivo. In addition, several additional fluorescent strains were created for future use (eed1∆-mCherry, cph1∆/efg1∆-mCherry, hgc1∆-mCherry, ras1∆-mCherry), all derived from previously described C. albicans mutants defective in the yeast-hyphae-transition.

An intravital microscopy model of the murine liver was adapted to allow for the observation of fungal cells within the organ. Parameters such as the age of the animals were found to severely reduce the quality of the images obtained, and hence the analysis of the colonization steps. After these improvements, fungal cells can now be observed reliably and reproducibly in the organ using our intravital microscopy system.

Starting with killed C. albicans yeasts, we observed that the temporal kinetics of fungal attachment to the liver sinusoids is significantly faster than expected. The process starts just seconds after injection of the fungi into the bloodstream, and is largely completed after only a few minutes. Especially the lateral, narrow sinusoids seem to be a preferred site of attachment. In many ways, this resembles the ad hoc adhesion to epithelial cells observed in vitro. Data acquisition is ongoing, and first steps have been taken to add a semi-automated image recognition software to aid in evaluating the time series obtained from the organ. Work planned for the near future includes investigating the early phase of yeast-hyphae-transition in vivo, and following the early immune response by additional staining of immune effector cells.

Selected publications

Brunke S, Hube B (2013) Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiol. 15(5):701-8

Lüttich A, Brunke S, Hube B, Jacobsen ID (2013) Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney. PloS one 8: e64482

Martin R, Albrecht-Eckardt D, Brunke S, Hube B, Hünniger K, Kurzai O (2013) A Core Filamentation Response Network in Candida albicans Is Restricted to Eight Genes. PloS one 8: e58613

Mayer FL, Wilson D, Hube B (2013) Candida albicans pathogenicity mechanisms. Virulence 4: 119-128

Mayer FL, Wilson D, Hube B (2013) Hsp21 potentiates antifungal drug tolerance in Candida albicans. PloS one 8: e60417

Miramon P, Kasper L, Hube B (2013) Thriving within the host: Candida spp. interactions with phagocytic cells. Med Microbiol Immunol 202: 183-195

Pietrella D, Pandey N, Gabrielli E, Pericolini E, Perito S, Kasper L, Bistoni F, Cassone A, Hube B, Vecchiarelli A. (2013) Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome. Eur J Immunol. 43: 679-692

Citiulo F, Jacobsen ID, Miramon P, Schild L, Brunke S, Zipfel P, Brock M, Hube B, Wilson D (2012) Candida albicans scavenges host zinc via Pra1 during endothelial invasion. PLoS Pathog 8: e1002777

Gow NA, Hube B (2012) Importance of the Candida albicans cell wall during commensalism and infection. Current Opinion in Microbiology 15: 406-412

Jacobsen ID, Wilson D, Wächtler B, Brunke S, Naglik JR, Hube B (2012) Candida albicans dimorphism as a therapeutic target. Expert Rev Anti Infect Ther. 10(1):85-93

Lüttich A, Brunke S, Hube B. (2012) Isolation and amplification of fungal RNA for microarray analysis from host samples. Methods Mol Biol. 845: 411-21

Mayer FL, Wilson D, Jacobsen ID, Miramon P, Slesiona S, Bohovych IM, Brown AJ, Hube B (2012) Small but crucial: the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans. PloS one 7: e38584

Miramón P, Dunker C, Windecker H, Bohovych IM, Brown AJ, Kurzai O, Hube B. (2012) Cellular Responses of Candida albicans to Phagocytosis and the Extracellular Activities of Neutrophils Are Critical to Counteract Carbohydrate Starvation, Oxidative and Nitrosative Stress. PLoS One 7(12):e52850.

Contact

Prof. Bernhard Hube

Tel. + 49 (0)3641 - 532 1401 oder 00

Abteilung Mikrobielle Pathogenitätsmechanismen
Leibniz Institut für Naturstoff-Forschung und Infektionsbiologie e.V.
Hans-Knöll-Institut (HKI)
Beutenbergstraße 11a
07745 Jena

Website of the Department of Microbial Pathogenicity Mechanisms (MPM) at the HKI

CEA family receptors: a novel class of immuno-regulatory proteins interacting with Candida albicans
E. Klaile

G-CSF revisited - Dual role of LBP in SIRS and sepsis Opposing function of LBP
H. Fang

Heme, Heme Degradation Products and Systems Biology of Sepsis-associated Organ Failure

Acronym: HHDP

Principal Investigator: Prof. Dr. Michael Bauer

Team: Dr. rer. med. Marcel Kramer, Peter Recknagel, Bianka Wissuwa, Beate Szafranski, Monique Riedel

Research Area: D Sepsis related Organ Failure

Project Number: D1.2

Duration: 01.08.2010 bis 31.07.2015

Module: In-house Professorship Group

The Problem

Hepatic dysfunction characterized by jaundice is traditionally viewed as a late feature of sepsis and portends poor outcome. The project aims to unravel molecular mechanisms of liver failure as well as its interaction with the heme degradation pathway.

Results so far

We hypothesized that changes in liver function occur early yet pass undetected by standard laboratory tests. Using long-term rat models of faecal peritonitis, predicted nonsurvivors displayed downregulation of genes encoding for proteins involved in phase I and II metabolism, and hepatobiliary transport. Simultaneously, both HMOX-1 and PI3K signaling were induced as early as six hours. Functional changes at fifteen hours included hepatocellular accumulation of bilirubin, bile acids and xenobiotics, with disturbed bile acid conjugation and drug metabolism. A comparable pattern of unconjugated and conjugated bile acids was also observed in plasma of patients fulfilling ACCP/SCCM criteria for severe sepsis on the day of diagnosis. In experimental animals cholestasis was preceded by loss of microvilli and internalization of the multidrug resistance-associated protein (Mrp2)-dependent transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi and Mrp2 in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. Liver dysfunction is thus an early event in sepsis, in which PI3K signaling plays a crucial role. All aspects of hepatic biotransformation are affected with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reᴀected by early alterations in plasma conjugated and unconjugated bile acids. These observations carry important implications for diagnosis, monitoring and pharmacotherapy in the critically ill.

To find genetic polymorphisms associated with human sepsis we screened genes involved in the heme degradation pathway in patients with severe human sepsis. We found a single nucleotide polymorphism rs2071746 and a GT-dinucleotide repeat in the promoter region of HMOX1, but no polymorphism in genes encoding biliverdin reductases (BLVRA and BLVRB) associated with the outcome of severe human sepsis. The functional impact of polymorphisms in the HMOX1 locus on sepsis outcome is far from being understood, but the polymorphisms potentially correlate with HO-1 expression. A correlation of the GT-dinucleotide repeat with HO-1 mRNA and protein levels has already been shown. Therefore, we analyzed the HMOX1 gene structure in more detail by sequencing HMOX1 transcripts. We were able to detect various alternatively spliced transcripts including a novel first exon in the 5` untranslated region (UTR) extending the current gene model. The evaluation of the alternative 5’-UTR splicing in the context of polymorphisms associated with sepsis outcome was done by a minigene approach. We found a dependency of splice-isoform frequencies and GT-dinucleotide repeat length. Since alternative 5’-UTR splicing is known to be involved in translation regulation we are currently validating the novel 5’-UTR transcripts in Luciferase reporter assays. This may provide a link between the genetic features of HMOX1, regulation of HO-1 activity and outcome.

Publications

Gonnert FA, Kunisch E, Gajda M, Lambeck S, Weber M, Claus RA, Bauer M, Kinne RW: Hepatic Fibrosis in a Long-Term Murine Model of Sepsis. Shock 2012 Apr, 37(4):399-407.

Gonnert FA, Recknagel P, Seidel M, Jbeily N, Dahlke K, Bockmeyer CL, Winning J, Lösche W, Claus RA, Bauer M: Characteristics of clinical sepsis reflected in a reliable and reproducible rodent sepsis model. J Surg Res 2011 Sep, 170(1):e123-34.

Kleiman A, Huebner S, Rodriguez Parkitna JM, Neumann A, Hofer S, Weigand MA, Bauer M, Schmid W, Schuetz G, Libert C, Reichardt HM, Tuckermann JP: Glucocorticoid receptor dimerization is required for survival in septic shock via suppression of interleukin-1 in macrophages. FASEB J. 2012 Feb;26(2):722-9.

Recknagel P, Claus RA, Neugebauer U, Bauer M, Gonnert FA: In vivo imaging of hepatic excretory function in the rat by fluorescence microscopy. J Biophotonics. 2012 Jan 23, [Epub ahead of print]

Recknagel P,  Gonnert FA, Westermann M., Lambeck S, Lupp A, Rudiger A, Dyson A, Carré JE, Kortgen A, Krafft C, Popp J, Sponholz C, Fuhrmann V, Hilger I, Claus RA, Riedemann NC, Wetzker R, Singer M, Trauner M, Bauer M:  Liver Dysfunction and Phosphatidylinositol-3-kinase Signaling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis. PLoS Medicine, 2012 accepted.

Sponholz C, Huse K, Kramer M, Giamarellos-Bourboulis EJ, Claus RA, Engel C, Kuhnt E, Kiehntopf M, Brunkhorst FM, Routsi C, Mylona V, Tsangaris I, Heinemann SH, Reinhart K, Platzer M, Bauer M: Gene polymorphisms in the heme degradation pathway and outcome of severe human sepsis. Shock in press

Weber M, Lambeck S, Ding N, Henken S, Kohl M, Deigner HP, Enot DP, Igwe EI, Frappart L, Kiehntopf M, Claus RA, Kamradt T, Weih D, Vodovotz Y, Briles DE, Ogunniyi AD, Paton JC, Maus UA, Bauer M: Hepatic induction of cholesterol biosynthesis reflects a remote adaptive response to pneumococcal pneumonia. FASEB J 2012 Jun, 26(6):2424-36.

Contact

Prof. Dr. Michael Bauer

Universitätsklinikum Jena
Klinik für Anästhesiologie und Intensivmedizin
Am Klinikum 1
07747 Jena

Role of HIF-1α and MORG1 in sepsis induced renal injury

Acronym: HIFMORG

Principal Investigators:
Claudia Schindler, Katrin Schindler

Scientific Advisors:
Prof. Dr. Gunter Wolf, MHBA
Tzvetanka Bondeva

Research Area: D Sepsis related
                          Organ Failure

Project Number: D1.3

Duration: 01.08.2010 - 31.07.2013

Module: PhD Fellowhip

The Problem

Clinical studies demonstrated that sepsis is associated with a high coincidence of an acute renal failure. Moreover, a pre-existing kidney disease is a major risk for an acute kidney injury. In this context, the project aims to gain more insights into the mechanisms of kidney dysfunction and whether Morg1 down-regulation or prolyl hydroxylases (PHDs) inhibition could reduce the severity of renal injury in sepsis via up-regulation of HIF-1α.

Results so far

The complex function of Morg1, HIF-1α and PHD3 in sepsis was studied by two mouse sepsis models CLP (cecal ligation and punture) and PCI (peritoneal contamination and infection) or LPS treatment in wild type (WT) (C57/Bl6) mice, Morg1 heterozygous (Morg1 HZ) mice and WT mice treated with PHD-Inhibitor (PHD-I) prior sepsis induction or LPS injection. The kidney damage after sepsis induction, or LPS injections was evaluated via analysis of the renal retention markers NGAL (neutrophil gelatinase-associated lipocalin) in blood plasma or urinary ACR. The alterations of NGAL values in blood plasma after 24 h in the experimental animals were significantly elevated in all models and a protective effect of PHD-I treatment or Morg1 HZ mice was not detected. On the other hand, analysis of the urinary ACR (albumin-creatinine ratio), another marker used to analyze the kidney function, showed no significant changes between the controls and treated mice in all models. This observation demonstrates the importance of the functional assays used to evaluate the kidney function in sepsis and its implications. The renal damage was also investigated via PAS-staining on kidney sections. Morg1 HZ and PHD-I pre-treated mice showed less damage of the kidney tissue upon sepsis induction, characterized with a reduced dilation and vacuolisation of proximal tubular cells compare with the WT septic mice. Especially in CLP-sepsis models the differences in the PAS-staining were obvious. Detection of the activated Caspase-3 by IHC on kidney sections demonstrated as well that in kidneys of Morg1 HZ and PHD-I treated mice were observed less apoptotic cells after sepsis induction or LPS injection then in WT animals.

IHC analysis found that the protein levels of HIF-1α, HIF-2α and TNFα were higher in Morg1 HZ sham and PHD-I treated sham mice compare with the WT sham animals. Furthermore, in CLP but not in PCI septic mice the protein levels of HIF-1α, HIF-2α, and TNFα were as well up-regulated in treated Morg1 HZ mice and PHD-I pre-treated mice relative to WT. Survival studies unveiled that neither Morg1 HZ mice, nor PHD-I treated mice were protected in CLP or PCI sepsis models compare with the WT septic mice. Nevertheless, the clinical severity score analysis (CSS) demonstrated that there was not a significant difference between the Morg1 HZ controls (sham operated) mice and CLP treated mice 24 h post CLP sepsis induction. Similarly, in PCI sepsis model, both Morg1 HZ and PHD-I treated mice demonstrated better conditions after 24 h PCI treatment, relatively to the WT mice. The LPS mouse model was in general less severe then CLP and PCI- sepsis models, and PHD-I treated mice showed lower CSS after 24 h LPS exposure in comparison to WT and Morg1 HZ animals. Our data demonstrate that the stimulation of HIF-1α has a nephroprotective function in the above experimental models.

Contact

Prof. Dr. Gunter Wolf, MHBA

Tel. +49 (0)3641 - 9 32 43 01

Universitätsklinikum Jena
Klinik für Innere Medizin III
Am Klinikum 1
07747 Jena

Recovery of energy metabolism in sepsis via leptin up-regulation by glucocorticoids as a new mechanism protecting from organ failure
A. Kleyman

Establishment of a Human Liver Model Supported by Microfluidics
A. Mosig

Sepsis-induced liver failure: Molecular mechanisms and functional significance of excretory dysfunction and impact on critical care pharmacology

Acronym: Liver Dysfunction

Principal Investigator: PD Dr. med. Andreas Kortgen

Team: Esther Fröhlich, Jaqueline Fischer

Research Area: D Sepsis related Organ Failure

Project Number: D1.11

Duration: 01.07.2011 bis 30.06.2015

Module: Senior Research Group

The Problem

The liver plays a key role in metabolism, excretion and host response in critically ill patients. Hepatic dysfunction in inflammation and sepsis is an early and regular feature with a profound impact on prognosis. The project aims to unravel molecular mechanisms of excretory hepatic dysfunction with impairment of transport mechanisms on the basolateral and canalicular membrane and the impact on critical care pharmacology.

Results so far

We hypothesized that hepatic dysfunction especially excretory dysfunction occurs early in the course of sepsis. Like ICG, DY635 is eliminated almost exclusively via hepatocytes into bile. However, kinetics of hepatocellular uptake and elimination are much faster. While in sham-operated rats fluorescence over the liver assessed with intravital microscopy decreases from its maximum value to 71±3% after 35 min for ICG, the value for DY635 is 17±4%. In septic rats (PCI model after 15 h) biliary excretion of the dyes is reduced. Fluorescence of DY635 after 35 minutes amounts to 36±6% of the peak value. Total amount of DY635 recovered in bile 1h after intravenous injection decreases from 60 % in sham animals to 34 % in septic animals. Gene expression of hepatocellular basolateral transporters Oatp1, Oatp2, Oatp8 und Ntcp responsible for uptake of endogenous and exogenous substances is significantly down-regulated in septic rats 15 h after induction of sepsis (PCI model) when compared to sham animals. At the canalicular pole of the hepatocyte gene expression of Mrp2, Mdr2 und Bsep is reduced, while Mdr1 is upregulated.

In 46 liver transplant recipients 12h after transplantation assessment of PDRICG was performed, accompanied by measurement of bile ICG concentration over 3h after iv-injection of the dye. In addition, plasma and bile concentrations of bilirubin were measured in the postoperative course. While PDRICG was not different with respect to patient and graft survival, peak ICG concentration and bile/plasma ratio of bilirubin on the first postoperative day were significantly reduced in non survivors and patients with necessity for retransplantation. Patients presenting sepsis and/or multi organ failure in the early postoperative course had significantly reduced values for peak bile ICG concentration and bile/plasma bilirubin ratio.

Contact

PD Dr. med. Andreas Kortgen

Tel.: +49 (0)3641 - 9 32 31 84

Universitätsklinikum Jena
Klinik für Anästhesiologie und Intensivmedizin
Am Klinikum 1
07747 Jena

Role of Nhe1 and Nkcc1 in the pathophysiology of sepsis
R. Mumtaz

AMPK associated insulin resistance in inflammation and sepsis
C. v. Löffelholz

Molecular Mechanisms of Candida Sepsis

Acronym: MomCanSep

Principal Investigator: Prof. Bernhard Hube

Team: Ilse Jacobsen, Betty Hebecker, Tony Pawlik, Nadja Jablonowski

Research Area: D Sepsis related Organ Failure

Project Number: D1.15

Duration: 01.08.2012 - 31.07.2015

Module: In-house Professorship Group

The aim of this project is to identify and characterize sepsis-associated fungal factors and their molecular interaction with host cells and thus to elucidate the molecular basis of Candida albicans sepsis

Project summary

Candida albicans is the most common cause of life-threatening nosocomial fungal infections. The mortality of sepsis caused by Candida is >50%,  higher than for bacterial septicaemias. During systemic infections, C. albicans can infect almost all organs, however, the host response and the ability to clear C. albicans cells is organ-specific. In the murine model of haematogenously disseminated candidiasis, which is the gold standard for investigating the pathogenesis of C. albicans bloodstream infections, mice die of progressive sepsis, kidney inflammation and failure, while fungal load declines in all other infected organs without causing pathological alterations. These data suggest that fungal pathogenicity is not only due to damage caused by the fungus, but also due to an inappropriate host response leading to damage caused by immune cells.

In this project, we aim to identify and characterise fungal factors which are responsible for the organ-specific host responses and the immunopathology in the kidneys. We propose that, (1) in addition to general cell wall components, distinct factors or activities are recognised and responsible for the differential host response and immunopathology; (2) fungal genes exist, which are not required for causing host damage, but for an organ-specific host response which is either protective or non-protective. We will use in vivo transcriptional profiling and large scale C. albicans mutant collection screening to identify such genes. Proof-of-principle candidate genes which have such characteristics have already been identified. These genes and their role during systemic C. albicans infection will be investigated in detail.

Therefore, this project aims at understanding the molecular mechanisms of sepsis induction triggered by C. albicans. The knowledge of the mechanisms responsible for sepsis caused by systemic candidiasis may provide potential targets for novel ways of immune therapy.

Work program

The work program comprises four main parts:

Part 1: Identification of stage-, tissue-, organ-, and infection-associated fungal genes potentially responsible for host responses

Part 2: Production of mutants lacking virulence associated genes and collection of large scale mutant libraries

Part 3: Large scale host response screening of mutant collections

Part 4: Detailed analysis of identified candidate genes for triggering sepsis

Vision

In this project, we aim at understanding the molecular mechanisms of sepsis induction triggered by C. albicans by identifying the fungal genes, factors or activities which are responsible for or contribute to the differential host responses in the different organs. Ultimately, the knowledge of the mechanisms responsible for sepsis caused by systemic candidiasis may provide potential targets for novel ways of immune therapy.

Selected publications

Seider K, Gerwien F, Kasper L, Allert S, Brunke S, Jablonowski N, Schwarzmüller T, Barz D, Rupp S, Kuchler K, Hube B (2014) Immune evasion, stress resistance, and efficient nutrient acquisition are crucial for intracellular survival of Candida glabrata within macrophages. Eukaryotic Cell 13:170-183

Brunke S, Hube B (2013) Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiol. 15(5):701-708

Lüttich A, Brunke S, Hube B, Jacobsen ID (2013) Serial passaging of Candida albicans in systemic murine infection suggests that the wild type strain SC5314 is well adapted to the murine kidney. PloS one 8: e64482

Martin R, Albrecht-Eckardt D, Brunke S, Hube B, Hünniger K, Kurzai O (2013) A Core Filamentation Response Network in Candida albicans Is Restricted to Eight Genes. PloS one 8: e58613

Mayer FL, Wilson D, Hube B (2013) Candida albicans pathogenicity mechanisms. Virulence 4: 119-128

Mayer FL, Wilson D, Hube B (2013) Hsp21 potentiates antifungal drug tolerance in Candida albicans. PloS one 8: e60417

Miramon P, Kasper L, Hube B (2013) Thriving within the host: Candida spp. interactions with phagocytic cells. Med Microbiol Immunol  202: 183-195

Pietrella D, Pandey N, Gabrielli E, Pericolini E, Perito S, Kasper L, Bistoni F, Cassone A, Hube B, Vecchiarelli A. (2013) Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome. Eur J Immunol. 43: 679-692

Citiulo F, Jacobsen ID, Miramon P, Schild L, Brunke S, Zipfel P, Brock M, Hube B, Wilson D (2012) Candida albicans scavenges host zinc via Pra1 during endothelial invasion. PLoS Pathog 8: e1002777 (→ Publication award of the DMyk)

Gow NA, Hube B (2012) Importance of the Candida albicans cell wall during commensalism and infection. Current Opinion in Microbiology 15: 406-412

Jacobsen ID, Wilson D, Wächtler B, Brunke S, Naglik JR, Hube B (2012) Candida albicans dimorphism as a therapeutic target. Expert Rev Anti Infect Ther. 10(1):85-93

Lüttich A, Brunke S, Hube B. (2012) Isolation and amplification of fungal RNA for microarray analysis from host samples. Methods Mol Biol. 845:411-21

Mayer FL, Wilson D, Jacobsen ID, Miramon P, Slesiona S, Bohovych IM, Brown AJ, Hube B (2012) Small but crucial: the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans. PloS one 7: e38584

Miramón P, Dunker C, Windecker H, Bohovych IM, Brown AJ, Kurzai O, Hube B. (2012) Cellular Responses of Candida albicans to Phagocytosis and the Extracellular Activities of Neutrophils Are Critical to Counteract Carbohydrate Starvation, Oxidative and Nitrosative Stress. PLoS One. 7(12):e52850

Contact 

Prof. Bernhard Hube

Tel. + 49 (0)3641 - 532 1401 oder 00

Abteilung Mikrobielle Pathogenitätsmechanismen
Leibniz Institut für Naturstoff-Forschung und Infektionsbiologie e.V.
Hans-Knöll-Institut (HKI)
Beutenbergstraße 11a
07745 Jena

Website of the Department of Microbial Pathogenicity Mechanisms (MPM) at the HKI

Dual role of PI3Kγ in the pathogenesis of septic cardiomyopathy

Acronym: PI3Kgamma

Principal Investigator: Bernadin Ndongson-Dongmo

Scientific Advisors: Prof. Reinhard Bauer, Prof. Reinhard Wetzker

Research Area: D Sepsis related Organ Failure

Project Number: D1.7

Duration: 01.04.2011 - 30.06.2014

Module: PhD Fellowship

The Problem

Cardiac performance during sepsis is impaired due to changes in the macro- and microcirculation, autonomic dysfunction, and inflammation-induced intrinsic myocardial depression. Signaling protein PI3Kγ negatively regulates contractility and relaxation via reduced cAMP availability by phosphodiesterase (PDE) activation preventing β-adrenergic overstimulation but stimulates extracellular remodeling and fibrosis. The project aims to unravel molecular mechanisms of the dual role of PI3Kγ in septic cardiomyopathy and will identify mechanisms for novel approaches in the management and treatment of sepsis-induced myocardial injury.

Results so far

We first searched for an in vivo phenotype in PI3Kγ-deἀcient mice and discovered during early period (3h) of LPS-induced SIRS (10 µg/g) a markedly increased contractility by 71±21%, whereas wildtype mice exhibited reduced myocardial contractility by 26±6% (P<0.05). This was accompanied by disturbed diastolic function in PI3Kγ-deficient mice 24 h after SIRS induction Measurements myocardial function were performed with the pressure-volume conductance catheter technique (MPCU-200™, Millar Instruments, Inc.). We hypothesize that these changes in myocardial function of PI3Kγ-deficient mice represent negative consequences of cardiomyocyte-disturbing muscular over-activation by uncontrolled β-adrenergic overstimulation because of the lack of PI3Kγ-mediated cAMP control in affected cardiomyocytes. Ongoing work aims to unravel if PI3Kγ-mediated β-adrenergic signaling by comparative analysis by PIP3 and/or PDE activation using knockin (PI3Kγ-kinase death) mice.

In addition, primary cell cultures of cardiomyocytes derived from adult mice was established ahead of schedule.

Publications

Schubert H, Eiselt M, Walter B, Fritz H, Brodhun M, Bauer R: Isoflurane/nitrous oxide anesthesia and stress-induced procedures enhance neuroapoptosis in intrauterine growth-restricted piglets. Intensive Care Med 2012, 38:1205-1214.

Richter F, Bauer R, Ebersberger A, Lehmenkuhler A, Schaible HG: Enhanced neuronal excitability in adult rat brainstem causes widespread repetitive brainstem depolarizations with cardiovascular consequences. J Cereb Blood Flow Metab 2012 Mar 28 [Epub ahead of print].

Contact 

Prof. Reinhard Bauer

Tel.: +49 (0)3641 - 9 39 56 36

Universitätsklinikum Jena
Institut für Molekulare Zellbiologie
CMB-Center for Molecular Biomedicine
Hans-Knöll-Straße 2
07745 Jena

Modification of gut barrier dysfunction in sepsis by phosphytidylcholine

Acronym: Phosphatidylcholine

Principal Investigator: Dr. Carsten Schmidt

Team: Dr. Philipp Reuken, Prof. Hortense Slevogt, Ursula Frischmann, Christian Lautenschläger

Research Area: D Sepsis related Organ Failure

Project Number: D2.1

Duration: 01.01.2012 - 31.12.2013

Module: Senior Research Group

The Problem

Gut barrier dysfunction gives rise to bacterial translocation and is one of the main causes of sepsis. We suggest that this barrier defect caused by mucus phosphatidylcholine (PC) deficiency can be reverted at least in part by oral substitution of delayed released PC. The project aims to investigate these prophylactic properties in a mouse model of sepsis.

Results so far

A significant reduction in mortality of sepsis can be reached mainly by prevention of sepsis induction and modulation of septic pathophysiology. Disturbances of microcirculation of the gastrointestinal tract give rise to bacterial translocation and are one of the main causes of sepsis. Recently, we were able to show that probe-based confocal laser endomicroscopy is able to quantify microcirculatory alterations in the gastrointestinal mucosa in a porcine model of septic shock. These results demonstrate a reduced vascularisation of the upper and lower GI-tract, preliminary data in patients suffering from sepsis show analogue results. Pathogenesis and supportive therapeutic implications of these sepsis related aspects are comparable in some aspects to ulcerative colitis, where three independent studies showed clinical response or remission of the disease in about 50% of patients. In prior studies of LPS-induced rodent sepsis an increased degradation of the extracellular protective phospholipid layer has been demonstrated, leading to an increased GI permeability and inflammation. We suggest that this barrier defect caused by mucus phosphatidylcholine deficiency can be reverted at least in part by oral substitution of delayed released PC compared to placebo in a murine sepsis model. Microbial translocation into the systemic circulation and affected organs are supposed to be reduced by PC-pretreatment. Moreover, outcome parameters such as morbidity and mortality as well as histological damage of affected organs will be examined. This concept will be followed by examination of local mucus PC content and hydrophobicity of the mucus (surface tension).

Contact

Dr. Carsten Schmidt

Tel. +49 (0)3641 - 9 32 46 44

Universitätsklinikum Jena
Klinik für Innere Medizin II
Abteilung Gastroenterologie, Hepatologie, Infektiologie
Am Klinikum 1
07747 Jena

Hepatic stellate cell activation during pneumonia
B. Giszas, R. Claus

A prospective, multi-center, randomized, double-blinded, placebo-controlled study for the evaluation of lloprost in the early postoperative period after liver transplantation
F. Rauchfuß

Mechanisms promoting and resolving - acute kidney injury in sepsis
G. Otto

Role of reactive oxygen species (ROS) in septic myocardial dysfunction

Acronym: Rosisep

Principal Investigator: Dr. med. Andreas Finkensieper

Team: Heike Fischer, Maria Wartenberg, Saskia Nitza

Research Area: D Sepsis related Organ Failure

Project Number: D1.8

Duration: 01.04.2011 - 31.03.2013

Module: Rotational Position

The Problem

Myocardial dysfunction frequently accompanies severe sepsis and septic shock and is known as septic cardiomyopathy. The management of sepsis-induced myocardial dysfunction remains supportive. Hence it is necessary to increase the knowledge of the pathophysiological as well as the molecular mechanisms which induce septic cardiomyopathy and to develop effcient novel approaches in treatment of septic myocardial dysfunction.

Results so far

The objective of this project is that septic plasma components (cytokines and others) induce an imbalance of reactive oxygen species (ROS) and nitric oxide (NO) in cardiomyocytes and these effects potential control myocardial dysfunction and viability. We postulate that an inhibition of critical members of the NADPH oxidase family may protect cardiomyocytes during sepsis and attenuate the inflammatory process and circumvent septic myocardial dysfunction as well.

Until now our team found a significant up-regulation of the NOX2 enzyme and a parallel down-regulation of the NOS2 enzyme in heart from mice 6 hours post induction of sepsis using the PCI model which was previously standardized by the CSCC. We postulate that these findings could be responsible for an imbalance of ROS and NO. Furthermore, we found an increased amount of Interleukin-1β in serum of septic mice 6 hours post induction of sepsis. In parallel, an up-regulation of transcripts of the Interleukin-1β receptor in the heart of mice derived from septic mice 6 hours post sepsis induction was ascertained. Therefore we propose an Interleukin-1β mediated pathway as one potential way how the expression of NOX2 and NOS2 could be regulated. In parallel, we found an increased ROS generation of beating cluster of cardiomyocytes differentiated from murine ES cells after exposure to serum derived from mice 6 hours post sepsis induction. Furthermore, the analysis of protein obtained from the heart of septic mice 6 hours post sepsis induction showed an increased activity of specific Matrix-Metalloproteases (MMPs). In parallel, an increased mRNA expression of MMP1, 3 and 9 was observed in heart of septic mice.

In conclusion, we postulate an important role of ROS mainly as second messenger during the development of septic myocardial dysfunction in combination with an increased activity of speciἀc MMPs.

Contact

Dr. med. Andreas Finkensieper

Tel. +49 (0)3641 - 9 32 41 39

Universitätsklinikum Jena
Universitäts-Herzzentrum
Klinik für Innere Medizin I
Am Klinikum 1
07747 Jena

Diagnostic risk assessment and therapeutic modulation of local and systemic sphingosine 1-phosphate (S1P) concen­trations and S1P-receptors for sepsis control
M. Gräler

Regulation of sepsis-induced endothelial cell dysfunction by 5’ AMP-activated protein kinase (AMPK)

Acronym: Seda

Principal Investigator: Silke Lindenmüller 

Scientific Advisor: Prof. Dr. Regine Heller

Research Area: D Sepsis related Organ Failure

Project Number: D1.12

Duration: 01.10.2011 - 30.09.2014

Module: PhD Fellowship

The Problem

The energy-sensing enzyme AMP-activated kinase (AMPK) is involved in the regulation of energy supply and stress protection in endothelial cells. The present project is aimed at investigating whether AMPK is modulated by inᴀammatory stimuli known to mediate sepsis and whether this enzymes is able to limit inflammatory responses of the endothelium and the development of endothelial dysfunction under septic conditions.

Results so far

In the first part of this project, the influence of inflammatory stimuli on AMPK activity was investigated (lipopolysaccharide (LPS), interleukin 1β (IL1β), interleukin 6 (IL6), interferon γ (IFNγ), tumor necrosis factor α (TNFα), alone or in combination). None of the tested stimuli led to an activation of AMPK as monitored by phosphorylation of the threonine 172 residue and by activity assays. In contrast, a phosphorylation at serine residue 485 was detected, but its function still needs to be studied. Interestingly, downregulation of the catalytic subunit AMPKα1 and/or α2 led to a decreased cytokine-induced expression of the adhesion molecules ICAM-1 and VCAM-1 indicating a decreased endothelial adhesivity. Our preliminary data suggest, that inhibition of AMPK by cytokines may help to limit the inflammatory response. This is in contrast to our initial hypothesis and needs further investigation with respect to the time-dependence of the effect and underlying mechanisms.

Contact

Silke Lindenmueller

Tel.: +49 (0)3641 - 9 39 56 05

Prof. Dr. Regine Heller

Tel.: +49 (0)3641 - 9 39 56 33

Universitätsklinikum Jena
Institut für Molekulare Zellbiologie, CMB
Hans-Knöll-Str. 2
07745 Jena

Establishing a molecular sensor for detection and treatment of sepsis
R. König

Protein acetylation in sepsis - modulation by inhibitors of protein deacetylases (HDACi)

Acronym: Sephi

Principal Investigator: Katrin Noack

Scientific Advisors: Prof. Dr. T. Heinzel, PD Dr. O.H. Krämer

Research Ares: D Sepsis related Organ Failure

Project Number: D1.13

Duration: 05.10.2011 - 04.10.2014

Module: PhD Fellowship

The Problem

Animal experiments showed that histondeacetylase inhibitors (HDACi) have a positive effect of the sepsis outcome and survival of mice. The project aims to investigate molecular mechanisms and possible targets of the HDACi as well as identifying the involved HDACs.

Results so far

We analyzed the influence of HDACi on IL6 secretion after stimulation of mouse embryonic fibroblasts (MEF) with (WT) and without p53. ELISA-Tests showed that IL6 was secreted by both cell lines after LPS stimulation, while WT MEFs secreted more IL6 than MEFs lacking p53, indicating a role of p53 in IL6 secretion. After treatment with VPA, IL6 levels after short time incubation with LPS in WT-MEF-cells were very slightly increased, but after long time exposure to LPS there was almost no difference between the cells with and without VPA detectable. Further Western Blot analysis showed that NFκB was activated through LPS: after short time stimulation activating phosphorylation of NFκB could be detected. Still, activation of NFκB does not lead to transcriptional activation of NFκB-target genes associated with apoptosis. Therefore other NFκB-target genes might be responsible for the IL6 secretion.

Contact 

Katrin Noack

Tel.: +49 (0)3641 - 9 49 354

Friedrich-Schiller-Universität Jena
Institut für Biochemie und Biophysik, CMB
Hans-Knöll-Straße 2
07745 Jena

Analysis of the mechanisms of chronification in a sepsis model in a cell- or organ-specific smad2 K.O. mouse

Acronym: SMAD2

Principal Investigator: Dr. rer. nat. Elke Kunisch 

Team: Prof. Dr. med. Raimund W. Kinne

research Area: D Sepsis related Organ Failure

Project Number: D1.4

Duration: 01.08.2010 - 31.07.2012

Module: Rotational Position

The Problem

Currently, nearly nothing is known about organ failure as long-term sequelae of sepsis. The project aims at characterizing the molecular mechanisms of long-term sequelae in a mouse model of sepsis.

Results obtained during the funding period

In collaboration with the CSCC, the Department of Anestesiology and the Institute of Pathology (Jena University Hospital), a long-term murine model of sepsis has been described by our group. The model showed a survival rate of approx. 40%. Whereas clinical severity score, food-intake, and body weight normalized until day 6, 4-week survivors still had an elevated white blood cell and platelet count, as well as elevated levels of markers for liver injury. In the liver of the mice, inflammatory infiltrates and substantial fibrosis was observed. Increased numbers of potentially pathogenetic macrophages and α-SMA-positive cells were detected in the vicinity of fibrotic areas. Together with augmented TGF-ß mRNA expression, increased phosphorylation of its signalling molecule Smad2 was seen. In order to address the role of the pro-fibrotic TGF-ß signalling for organ-fibrosis in sepsis, a fibroblast-specific Smad2 k.o. mouse was established. Initial short-time sepsis trials showed a higher survival rate in fibroblast-specific Smad2 k.o. mice (100%) than in wild-type mice 24 h after sepsis-induction (63%).

Publications

Gonnert FA, Kunisch E, Gajda M, Lambeck S, Weber M, Claus RA, Bauer M, Kinne RW: Hepatic Fibrosis in a Long-Term Murine Model of Sepsis. Shock 2012 Apr, 37(4):399-407

Contact 

Prof. Dr. med. Raimund W. Kinne

Tel.: +49 (0)36691 - 81 228

Universitätsklinikum Jena
Lehrstuhl für Orthopädie
AG Experimentelle Rheumatologie
Klosterlausnitzer Str. 81
07607 Eisenberg

The somatostatin receptor 2A as a possible therapeutic target in sepsis and associated liver-dysfunction

Acronym: SomatostatinR

Principal Investigator: Dipl. Biol. Isabell Lenhardt 

Scientific Advisor: apl. Prof. Dr. med. habil. Amelie Lupp

Research Area: D Sepsis related Organ Failure

Project Number: D1.6

Duration: 01.03.2011 - 28.02.2014

Module: PhD Fellowship

The Problem

About 20 to 25% of patients with sepsis and associated organ failure quite early display an impaired liver function, which in severe cases can result in acute liver failure. However, despite extensive research efforts no specific therapy exists so far for the treatment of liver failure in sepsis. Since literature data suggest an immunomodulatory action of somatostatin, the aim of our investigations is to elucidate whether somatostatin receptors (especially the somatostatin receptor 2A [SSTR2 A]) can serve as possible targets for the treatment of liver dysfunction in sepsis e.g. using long-acting somatostatin analogues.

Results so far

Using two different models for the induction of a systemic inflammatory reaction in mice, intraperitoneal administration of lipopolysaccharides (LPS) and peritoneal contamination and infection with human stool bacteria (PCI), it could be shown that SSTR2A knockout mice are more susceptible to the induction of a systemic inᴀammation than wild-type and heterozygous animals. These differences between the SSTR2A mouse genotypes were seen not only with respect to liver function and to the oxidative state in liver tissue but also with regard to other organ systems (lung, cardiovascular system, kidney), thus pointing to an involvement of the SSTR2A in systemic inᴀammatory reactions.

Further experiments carried out subsequently revealed (as expected), that a concomitant treatment of wild-type mice with the long-acting somatostatin analogue octreotide was able to reduce the LPS induced effects both in liver and in different other organ systems. In contrast (and as also expected), no protective effects were observed in SSTR2A knockout mice.

Contact 

apl. Prof. Dr. med. habil. Amelie Lupp

Tel. +49 (0)3641 - 9 32 56 78

Universitätsklinikum Jena
Institut für Pharmakologie und Toxikologie
Drackendorfer Str. 1
07747 Jena

Role of low-molecular-weight inhibitors of acid sphingomyelinase in sepsis-induced hepatic stellate cell activation
H. Chung, R. Claus

Role of the Lectin-like Domain of Thrombomodulin in Murine Sepsis-Induced Cardiac Depression

Acronym: Thrombomodulin

Principal Investigator: PD. Dr. med. Sebastian Stehr

Team: M. sc. Nadine Hechaichi, Dipl.-Ing. Jacqueline Fischer (FH)

Research Area: D Sepsis related Organ Failure

Project Number: D1.10

Duration: 01.07.2011 - 31.06.2014

Module: Senior Research Group

The Problem

Cardiac depression is observed as a feature of sepsis and is related with a poor outcome. The project aims to unravel molecular mechanisms of cardiac failure in dependence of Thrombomodulin as well as the interactions of Thrombomodulin with HMGB1 as a potential ligand.

Results so far

In preliminary experiments we saw a strong indication of a pivotal role of the lectin-like domain of thrombomodulin (LLD of TM) and its anti-inflammatory effects in sepsis. The results indicate an interaction between the LLD of TM and the alarmin HMGB1. We therefore hypothesize that the LLD of TM plays a central role in sepsis-induced cardiac depression by modulating HMGB1 bioavailability.

We hypothesize an increased inflammatory effect in mice lacking the LLD of TM as a result of increased HMGB1 signaling and therefore a reduced survival in mice with polymicrobial sepsis lacking the LLD of TM due to a reduction of anti-inflammatory effects. We expect this signaling cascade to involve the TLR2, TLR4 or RAGE pathway.

The siRNA knockdown of TLR2, TLR4 and RAGE will be used to evaluate respective effects on HMGB1 sequestration after stimulation in cell culture. The cell culture of primary cardiomyocytes was established and the completion of transcriptome-analysis after LPS-stimulation is in process. The establishment of a protocol for transfection of these cells with pcDNA of human full-length TM or pcDNA of human TM lacking the LLD and for knockdown the different receptor pathways via siRNA is ongoing. After stimulation with LPS, we hypothesize that these experiments will indicate a signaling cascade that is involved in the anti-inflammatory effect of the LLD of TM. Furthermore the metabolism of hearts from mice lacking the LLD of TM and wild type controls will be studied after sepsis in the isolated working heart. A murine isolated working heart model with possibilities of measuring glucose and fatty acid oxidation is available for the planned experiments. In future planned experiments, we will use in vivo anti-RAGE, -TLR2 or -TLR4 antibodies and in vivo hydro-dynamic over expression of the LLD of TM to evaluate a possible treatment for sepsis-induced myocardial depression.

Contact 

PD. Dr. med. Sebastian Stehr

Tel.: +49 (0)3641 - 9 32 31 46

Universitätsklinikum Jena
Klinik für Anästhesiologie und Intensivmedizin
Am Klinikum 1
07747 Jena

Visualization of Immune Cell Activation by Sepsis Pathogens using Life-Cell Imaging
O. Kurzai, F. Essig