Pia Ockelmann

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

Prednisolone as preservation additive prevents from ischemia reperfusion injury in a rat model of orthotopic lung transplantation.

The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.

Deguelin Attenuates Reperfusion Injury and Improves Outcome after Orthotopic Lung Transplantation in the Rat

The main goal of adequate organ preservation is to avoid further cellular metabolism during the phase of ischemia. However, modern preservation solutions do rarely achieve this target. In donor organs hypoxia and ischemia induce a broad spectrum of pathologic molecular mechanisms favoring primary graft dysfunction (PGD) after transplantation. Increased hypoxia-induced transcriptional activity leads to increased vascular permeability which in turn is the soil of a reperfusion edema and the enhancement of a pro-inflammatory response in the graft after reperfusion. We hypothesize that inhibition of the respiration chain in mitochondria and thus inhibition of the hypoxia induced mechanisms might reduce reperfusion edema and consecutively improve survival in vivo. In this study we demonstrate that the rotenoid Deguelin reduces the expression of hypoxia induced target genes, and especially VEGF-A, dose-dependently in hypoxic human lung derived cells. Furthermore, Deguelin significantly suppresses the mRNA expression of the HIF target genes VEGF-A, the pro-inflammatory CXCR4 and ICAM-1 in ischemic lungs vs. control lungs. After lung transplantation, the VEGF-A induced reperfusion-edema is significantly lower in Deguelin-treated animals than in controls. Deguelin-treated rats exhibit a significantly increased survival-rate after transplantation. Additionally, a downregulation of the pro-inflammatory molecules ICAM-1 and CXCR4 and an increase in the recruitment of immunomodulatory monocytes (CD163+ and CD68+) to the transplanted organ involving the IL4 pathway was observed. Therefore, we conclude that ischemic periods preceding reperfusion are mainly responsible for the increased vascular permeability via upregulation of VEGF. Together with this, the resulting endothelial dysfunction also enhances inflammation and consequently lung dysfunction. Deguelin significantly decreases a VEGF-A induced reperfusion edema, induces the recruitment of immunomodulatory monocytes and thus improves organ function and survival after lung transplantation by interfering with hypoxia induced signaling.

The fibrinopeptide bβ15-42 reduces inflammation in mice subjected to polymicrobial sepsis.

ABSTRACT Sepsis is still a leading cause of death on intensive care units. Despite intensive research, only few new therapies have been developed and used in the clinical setting. The fibrin fragment B&bgr;15-42 was already shown to preserve endothelial barrier function by binding to VE-cadherin and thus stabilize the interendothelial junctions. This was accompanied by reduced inflammation. Now we show that treatment with B&bgr;15-42 reduces inflammation in a murine polymicrobial sepsis model. Administration of B&bgr;15-42 reduced proinflammatory cytokine levels in the lung, liver, and blood and decreased neutrophil infiltration into the lung. Analysis alanine aminotransferase and aspartate aminotransferase further indicated reduced liver damage following polymicrobial sepsis. In vitro experiments using endothelial cells and macrophages further revealed that B&bgr;15-42 had no direct effect on Toll-like receptor–mediated inflammation. Therefore, we assume that attenuated inflammation is rather due to sustained vascular integrity and thus suppresses vascular leakage and subsequently leukocyte infiltration during sepsis.

Cecum ligation and dissection: a novel modified mouse sepsis model.

BACKGROUND The fact that many sepsis therapeutics failed to be translated into the human indicates that there is still a serious need to reassess our models of sepsis research. We aimed to develop a novel modified model of sepsis in the mouse, which simulates the clinical situation more accurately. MATERIALS AND METHODS Sepsis was induced in C57Bl/6 mice by dissecting the cecum and placing the discontinued organ back into the abdomen (cecum ligation and dissection [CLD]). Septic animals were relaparotomized after 6 h, followed by peritoneal lavage, and antibiotic treatment. Results were compared with shams or the classic colon ligation and puncture (CLP) model. The postoperative lung impairment was assessed using neutrophil invasion as a surrogate. Proinflammatory cytokines were measured by either real-time polymerase chain reaction or Luminex technology, and liver damage was evaluated by aspartate transaminase and alanine transaminase measurements. RESULTS In CLD animals with relaparotomy after 6 h, lung interleukin (IL) 6, monocyte chemoattractant protein (MCP)-1 messenger RNA levels, and neutrophil invasion were significantly increased. Liver messenger RNA expression in CLD animals was significantly upregulated for IL-6, tumor necrosis factor alpha, IL-10, and MCP-1 compared with sham and CLP animals. Significantly higher levels of alanine transaminase were observed in CLD animals. Finally, systemic inflammation as measured by plasma IL-6, tumor necrosis factor alpha, IL-1β, IL-10, and MCP-1 was significantly increased in all CLD animals compared with shams, whereas CLP animals only showed an insignificant increase in the latter molecules. CONCLUSIONS Our modifications to the classic CLP model significantly produced organ inflammation, liver damage, and a similar mortality compared with a clinical setting, with a reliable onset of sepsis.

VEGF-A Blockade Reduces Reperfusion Edema but Favors Arterial Thromboembolism in a Rat Model of Orthotopic Lung Transplantation:

Background Ischemia-reperfusion edema is a common early complication after lung transplantation where the hypoxia-induced vascular endothelial growth factor (VEGF)-A plays a pivotal role. It remains unclear whether a VEGF blockade is beneficial in lung transplantation. Methods VEGF-A blockade was investigated in an orthotopic rat model of lung transplantation. VEGF-A antibody was added into the preservation solution alone (&agr;-VEGF D/−), in the preservation solution and systemically to the recipient before reperfusion (&agr;-VEGF D/R), or applied to the recipient alone before reperfusion (&agr;-VEGF −/R). Forty-eight hours after lung transplantation, left lungs were collected and wet-to-dry ratio, Western blotting, RT-PCR, and immunohistology were performed. Results VEGF-A blockade in &agr;-VEGF D/−, &agr;-VEGF D/R, and &agr;-VEGF −/R resulted in neutralization of tissue VEGF-A. Reperfusion edema was only reduced in &agr;-VEGF D/R and &agr;-VEGF D/− groups versus Perfadex controls. Some &agr;-VEGF −/R rats showed a hyperinflammation leading to increased pro-inflammatory cytokine expressions as well as increased edema. Whereas generally the &agr;-VEGF D/− group showed decreased inflammation, the combination with anti-VEGF treatment to the recipient resulted in a pro-inflammatory and a pro-apoptotic phenotype. Short-term survival, however, was not significantly different in all groups as compared to the controls. In the &agr;-VEGF (D/R) or (D/−) groups, animals mainly died from arterial thromboembolisms and in the &agr;-VEGF (−/R) group, hyperinflammation was the main cause of death. Conclusion VEGF-A directly contributes to the formation of a reperfusion edema, which might be reduced by its blockade. However, the &agr;-VEGF effect on the endothelial integrity might also favor arterial thrombosis formation.

Abstract 4957: Post chemotherapy aggressiveness of breast cancer cells is related to TLR-3 / NOD-1 -2 activation.

Post chemotherapy relapsing breast cancer is often characterized by an increased aggressiveness and potential to metastasize. The mechanisms by which cancer cells escape chemotherapy are yet not completely understood. Toll-like receptor (TLR)-3, activated by RNAs, as well as NOD-1 and NOD-2, two newly described regulators of the innate immune system, play a role in tumor growth by regulating apoptosis or macrophage invasion into tumors. We hypothesize that chemotherapy leads to release of nucleotides from apoptotic cells which then in turn are capable of activating the pro-inflammatory TLR-3 pathway. The parallel activation of NOD-1/-2 via TRAF6 then leads to an amplification of the metastasizing potential of post chemotherapy breast cancer cells by increasing the recruitment of macrophages. Balb/c mice bearing 4T1 syngeneic breast cancer tumors were treated with a combination chemotherapy (CMF; cyclophosphamide 50 mg/kg, methotrexate 25 mg/kg, 5-fluoro-uracil 50 mg/kg; n=8) cycled twice i.p., solvent (NaCl; n=8) cycled twice i.p. or without therapy (n=8). After 21 days animals were sacrificed, tumor weight was measured, metastases were detected histologically, cell proliferation rate was analyzed by Ki-67 expression, macrophage count was evaluated immunohistologically and tissue mRNA expression was examined by RT-PCR. CMF treatment (376.0 ± 61.14 mg) significantly retarded tumor growth vs. untreated (620.6 ± 58.95 mg) or NaCl (665.8 ± 45.46 mg) treated controls (P TLR-3 activation in 4T1 breast cancer tumors might lead to increased NOD-1/ -2 activation and simultaneously to increased macrophage invasion representing an independent indicator for the aggressiveness of breast tumors. Supported by a grant from the Grants4Targets initiative from Bayer Pharma AG to P.P. Citation Format: Patrick Paulus, Peter Ellinghaus, Anja Urbschat, Karin Zins, Pia Ockelmann, Christin Reissig, Kai Zacharowski. Post chemotherapy aggressiveness of breast cancer cells is related to TLR-3 / NOD-1 -2 activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4957. doi:10.1158/1538-7445.AM2013-4957

Pssst ... AINS-Secrets! – Heute aus dem Bereich Intensivverlegung

Ring-Ring ... das Handy der jungen Assistenzärztin Dr. Valeska Schimanski zeigt eine neue SMS an: „Achtung an alle – Notarzt gesucht für einen Flug heute von Rumänien nach Schweden. Abfl ug 10:30 Uhr. Wer kann, bitte melden.“ Erst gestern hatte Dr. Schimanski sich auf www.I-need-aDoc.de regis triert – und schon heute hat sie die Möglichkeit, einen Rückholdienst zu fl iegen. Sie ruft schnell an und sichert sich den Flug. Schließlich ist das ja „einfach verdientes Geld“. Circa eine Stunde später sitzt sie in einem als Ambulanzjet eingerichteten Learjet und bereitet sich auf den Start vor. Die Turbinen heulen auf und es geht los. Beim Aufstieg verspürt sie einen starken Druck auf den Ohren, den sie mit Gähnen auszugleichen versucht.

Pssst ... AINS-Secrets! – Heute aus der Augenheilkunde

ASA-Klassifi kation Die Einteilung nach der American Society of Anaesthesiologists (ASA) geht auf das Jahr 1941 zurück. Ein Komitee der ASA beauftragte Saklad, Rovenstine und Taylor eine Kategorisierung anhand von statistischen Auswertungen zu fi nden, die eine perioperative Gruppierung von Patienten bezüglich ihres Operationsrisikos und ihrer Komplikationsrate ermöglichen sollte. Diese Aufgabe wurde von ihnen als nicht lösbar defi niert. Stattdessen entwickelten sie eine 6 PunkteSkala, die eine allgemeine Einstufung des präoperativen Patientenstatus ermöglichte. Sie hoff ten, dass die Anästhesisten Amerikas diese Klassifi kation übernehmen würden. Damit wäre eine statistische Auswertung der Daten bezüglich Letalität und Morbidität von operativen Prozeduren möglich. Die Klassen 1–4 ähnelten den Klassen 1–4 der heutigen Defi nition. In einer Notfallsituation wurden Patienten der Klasse 1–2 in Klasse 5 eingestuft. Vergleichbar wurden Patienten, die normalerweise als Klasse 3 oder 4 Fachwissen: AINS-Secrets

Pssst ... AINS-Secrets! – Heute aus der Intensivmedizin

Sonntag 03:30 Uhr: Der Bereitschaftsarzt der Anästhesie Olaf F. wird durch einen Reanimationsalarm aus dem Schlaf gerissen: „Station 5 leblose Person“. Rasch zieht er seine Kleidung an und hetzt zur Station. Ein 73-jähriger Patient liegt zyanotisch im Bett; die Schwester hat bereits mit den Reanimationsmaßnahmen begonnen. Sie berichtet, dass der Patient sie ins Zimmer gerufen habe und angab, starke Schmerzen in der Brust zu haben. Direkt danach sei er bewusstlos geworden. Dr. F. beginnt, den Patienten suffi zient mit Maske zu beatmen und lässt durch das nachfolgende AnästhesieTeam ein EKG und die Sauerstoff sättigung anschließen sowie die Intubation vorbereiten. Im EKG zeigt sich ein Kammerfl immern, das nach dem ersten Zyklus der Reanimationsmaßnahmen defi brilliert wird – zu einer Konversion kommt es jedoch nicht. Der Atemweg des Patienten soll nun mittels Tubus gesichert werden – doch bei der Einstellung mit dem Laryngoskop sieht Dr. F. nur die Epiglottis (Cormack und Lehane Grad III). Trotz Führungsstab gelingt es ihm nicht, den Tubus zu platzieren. Nach erneuter Beatmung und Optimierung der Lagerung im Sinne einer verbesserten JacksonPosition versucht er es ein 2. Mal und er kann den Tubus „blind“ problemlos platzieren. Bei direkt folgender Auskultation sind die Lungen frei und es ist ein gleichseitiges Atemgeräusch zu hören. Nach Gabe von 3 mg Adrenalin i. v. und erneuter Defi brillation konvertiert der Patient in einen tachykarden Sinusrhythmus. Nach weiterer Stabilisierung wird der Patient analgosediert auf die Intensivsta tion verlegt. Dort wird neben einem arteriellen Zugang auch ein Kühlkatheter zur Initiierung einer milden Hypothermie über 24 Stunden angelegt.