Alexandra Westerholt

Cecal ligation and puncture versus colon ascendens stent peritonitis: two distinct animal models for polymicrobial sepsis.

Colon ascendens stent peritonitis (CASP) and cecal ligation and puncture (CLP), two animal models designed to closely mimic the clinical course of intra-abdominal sepsis, were compared. In the past, immunomodulatory therapies developed in animal studies failed to be successful in humans. As a consequence, the established animal sepsis models were criticized. It has been proposed that present models had to be reevaluated, and new, clinically more relevant models should be evolved. CLP procedure was performed puncturing once (CLP[1]) or twice (CLP[2]) the ligated cecum of C57BL/6 mice. In the CASP model, a stent with defined diameter was surgically inserted into the ascending colon. Survival, bacterial load, immunohistochemistry, and serum cytokine levels were analyzed in the groups. Survival after CASP procedure correlated strongly with the stent diameter, whereas the number of punctures in CLP did not significantly change survival rate. Bacterial loads of peritoneal lavage, liver, and lung, as well as serum cytokine levels (tumor necrosis factor, interleukin 1β, interleukin 10) steadily increased from 6 to 24 h after the CASP procedure. In contrast, continuously low amounts of bacteria and cytokines were found in CLP mice at any point of time. Twenty-four hours after CLP surgery, the ligated cecum was covered by adhesive small bowel loops, whereas in CASP mice, the intestinal leakage was then still present. The CASP model mimics closely the clinical course of diffuse peritonitis with early and steadily increasing systemic infection and inflammation (systemic inflammatory response syndrome). In contrast, CLP reveals a model of intra-abdominal abscess formation with sustained and minor signs of systemic inflammation.

Impaired Monocyte IL-12 Production Before Surgery as a Predictive Factor for the Lethal Outcome of Postoperative Sepsis

ObjectiveTo investigate whether monocyte paralysis resistant to interferon-gamma (IFN-&ggr;) costimulation may exist before surgery and postoperative infection and may correlate with the outcome of postoperative sepsis. Summary Background DataSeveral studies have correlated monocyte paralysis during the course of sepsis with lethal outcome. Although the authors’ previous work indicated that preoperative defects in monocyte interleukin (IL)-12 production are associated with the development of severe postoperative sepsis, the functional state of monocytes before surgery and infection and its significance for sepsis requires further analysis. MethodsIn a prospective study, monocyte functions of 1,113 consecutive patients were examined before major visceral surgery. Monocytes were isolated from peripheral blood and were stimulated in vitro with IFN-&ggr; and lipopolysaccharide. The secretion of IL-12 p70, IL-12 p40, IL-10, and tumor necrosis factor was measured. ResultsPreoperative monocyte secretion of IL-12 p70 and IL-12 p40 was significantly reduced in patients who developed lethal postoperative sepsis compared with sepsis survivors and patients with uneventful postoperative recovery. Moreover, preoperative monocyte IL-12 production was an independent predictive factor for the lethal outcome of postoperative sepsis by multivariate analysis. Preoperative monocyte IL-10 production was impaired in the sepsis group but did not correlate with death from sepsis. Preoperative monocyte tumor necrosis factor secretion was comparable between patients with uneventful recovery, sepsis survivors, and nonsurvivors. Thus, impaired preoperative monocyte IL-12 secretion in patients developing lethal postoperative sepsis did not result from an overproduction of IL-10 or from a generalized monocyte paralysis. The association between impaired preoperative monocyte IL-12 production and death from sepsis was also not explained by gender differences, underlying malignant disease, tumor type, neoadjuvant therapy, or age. ConclusionsThese results identify a selective preoperative defect in monocyte IL-12 production as a predictive factor for the lethal outcome of postoperative sepsis. These data suggest that a partial preoperative monocyte paralysis severely impairs the host defense against postoperative infection, resulting in an increased risk of lethal sepsis.

Selective depletion of alveolar macrophages in polymicrobial sepsis increases lung injury, bacterial load and mortality but does not affect cytokine release.

Background: Resident tissue macrophages exert important functions during severe systemic infection and contribute to changes in local as well as systemic immune responses. Alveolar macrophages (AM) play a crucial role in airway diseases and in the defense against microorganisms invading the body via the bronchopulmonary tract. It has been postulated that AM are involved in the development of acute local disorders as a consequence of extrapulmonary stimuli like pancreatitis, peritonitis, or trauma. Objective: The aim of this study was to analyze the local and systemic role of AM during sepsis using selective AM depletion in the murine colon ascendens stent peritonitis (CASP) model of polymicrobial sepsis. Methods: 48 h prior to CASP surgery, AM of female C57BL/6 mice were selectively depleted by intratracheal application of clodronate liposomes (Lipo-clod). For control purposes, phosphate-buffered saline (PBS) liposomes (Lipo-PBS) were used. Results: CASP led to significantly elevated levels of local and systemic cytokines independent of the presence of AM. In contrast, levels of gut-derived bacteria in bronchoalveolar lavage and lung of septic mice were significantly higher in Lipo-clod-treated animals compared to Lipo-PBS-treated animals. After CASP-induced sepsis, local barrier dysfunction in the lung was detected; AM depletion resulted in severely enhanced development of acute lung injury. Consequently, Lipo-clod-treated animals showed strongly reduced survival rates after CASP. Conclusions: Contrarily to other macrophage populations, AM do not significantly contribute to local and systemic cytokine release during polymicrobial abdominal sepsis. AM have important protective functions for local clearance of gut-derived bacteria and attenuation of lung injury.

Importance of T cells to accelerated rejection and acceptance of renal allografts in sensitized rat recipients

BACKGROUND Sensitized recipients often experience fulminant allograft loss by yet ill-defined cellular and/or humoral immune mechanisms. In this study, we analyzed the contribution of cellular elements, in particular T cells, to the accelerated rejection of renal allografts in sensitized rats. METHODS AND RESULTS LEW rats sensitized with BN skin grafts died of uremia in 3.3+/-0.9 days after transplantation of a BN kidney, similarly to bilaterally nephrectomized animals. Adoptive transfer of 10(6) graft-infiltrating mononuclear cells as well as their CD25+ subset into otherwise normal LEW recipients accelerated rejection of BN test cardiac allografts (5.4+/-0.5 days to 6.6+/-0.4 days vs. 7.8+/-0.8 days in controls, P<0.0007), while the CD25- population was ineffective (8.0+/-0.6 days, NS). Furthermore, alpha/beta-T-cell receptor (TCR)-targeted therapy with R73 monoclonal antibody abrogated accelerated rejection, and produced long-term survival in sensitized animals treated before kidney engraftment (day -7 to day -1). Long-term survival was associated with an up-regulation of intragraft interleukin-4 and interleukin-10 expression in conjunction with depressed Th-1-type cytokines. In addition, alpha/beta-TCR-targeted therapy even in low subtherapeutic dose decreased IgM alloantibody levels, and prevented the switch from IgM to IgG alloantibody response. CONCLUSIONS This is the first report that documents the striking efficacy of alpha/beta-TCR-targeted therapy in sensitized rat renal transplant recipients. The results provide evidence for a critical role of T cells for both accelerated rejection and long-term graft survival. Up-regulation of Th2-type cytokine profile may, at least in part, contribute to the acquisition of immune unresponsiveness after alpha/beta-TCR-targeted therapy in this well-defined rat renal transplant model.

Management bei abdomineller Sepsis

Management of Abdominal Sepsis Sepsis is still a major problem and accompanied by a high perioperative morbidity and mortality in surgical intensive care units. It is a clinical syndrome that complicates severe local abdominal infection (peritonitis) and is characterized by systemic inflammation and widespread extraperitoneal systemic tissue injury, requiring multidisciplinary coordination of intensive care. Crucial is rapid diagnosis and an early goaldirected causal therapy of abdominal sepsis in terms of radical focus sanitation as well as aggressive systemic antibiotics. These standard therapy regimes are supplemented by additional therapeutic strategies depending on the severity score.

Komplikationsmanagement nach Ösophagektomie

Die gravierendste Komplikation nach Ösophagektomie, eine Sepsis infolge einer Mediastinitis auf dem Boden einer Anastomoseninsuffizienz, ist trotz moderner Intensivtherapie weiterhin mit einer hohen Morbidität und Mortalität verbunden. Neben der mediastinalen Manifestation weitet sich das Krankheitsbild rasch im Sinne einer Sepsis auf den gesamten Körper aus. Wie bei jedem septischen Krankheitsbild entscheidet die unverzügliche Diagnosestellung mit unmittelbarer Therapieeinleitung über die Prognose. Die kausale Therapie besteht zuallererst in einer frühzeitigen interventionellen bzw. chirurgischen Herdsanierung sowie in einer sofortigen breiten, später gezielten antimikrobiellen Behandlung. Bei den ersten Sepsiszeichen postoperativ (Verschlechterung des Allgemeinzustands, verzögerte postoperative Rekonvaleszenz, systemische Entzündungszeichen, suspekter abdomineller Untersuchungsbefund, auffällige Drainagesekretion) sollte nach Ösophagektomie immer zuerst an eine Mediastinitis als Folge einer Anastomoseninsuffizienz gedacht bzw. diese ausgeschlossen werden. Der erste diagnostische Schritt besteht in der Regel in der endoskopischen Kontrolle der Anastomose. Dabei sollte neben einer möglichen Leckage vor allem die Interponatdurchblutung beurteilt werden. Die therapeutischen Möglichkeiten umfassen je nach Lokalisation und klinischer Klassifikation der Insuffizienz eine einfache Drainageableitung, eine zusätzliche endoskopische Intervention meist mit Stentplatzierung sowie eine Reoperation bis hin zur Diskontinuitätsresektion. Die Säulen der Sepsistherapie – rascher Therapiebeginn, radikale Herdsanierung, unverzügliche Applikation von Breitbandantibiotika und der Einsatz moderner intensivtherapeutischer Konzepte – sind die Basis für ein erfolgreiches postoperatives Komplikationsmanagement nach Ösophagektomie und entscheiden über die weitere Prognose des Patienten.

#73 Protection from severe chronic stress-induced immune depression and sickness behavior by inhibition of indoleamine-2,3-dioxygenase

depression and sickness behavior by inhibition of indoleamine-2,3-dioxygenase Cornelia Kiank , Jan-Philip Zeden , GerhardFusch ,Nam T. Nguyen , Astrid Starke , Alice Mundt , Alexandra Westerholt , Johannes-Peter Haas , Christine Schutt a a Department of Immunology, University of Greifswald b Department of Surgery, University of Greifswald c Department of Pediatrics, Division of Neonatology and Pedriatic Intensive Care, University of Greifswald

131 Acute stress is followed by enhanced tryptophan catabolism

Astrid Starke , Nam T. Nguyen , Jan-Philip Zeden , Cornelia Kiank , Georg Daeschlein , Gerhard Fusch , Alice Mundt , Johannes-Peter Haas , Alexandra Westerholt , Christine Schutt a a Department of Immunology, University of Greifswald, Germany b Department of Surgery, University of Greifswald, Germany c Department of Pediatrics, Division of Neonatology and Pedriatic Intensive Care, University of Greifswald, Germany d Institute of Hygiene and Environmental Medicine, University of Greifswald, Germany

Protektive Funktion von NK1.1 + Zellen in der murinen polymikrobiellen Peritonitis

Abdominal sepsis due to anastomosis insufficiency after major abdominal surgery remains a life threatening condition. Interferon-gamma (IFNγ) is rapidly upregulated after inflammatory stimuli and is known to be a potent inducer of bactericidal effector mechanisms. Clinical as well as experimental data point to an essential protective role of IFNγ in the course of abdominal sepsis. The cellular source of IFNγ, especially at early stages of sepsis remains unclear. T cells can secrete substantial amounts of IFNγ but therefore require time for activation, maturation, and clonal proliferation. Thus, NK cells (or NKT cells) are better candidates for early IFNγ production in vivo. Our aim was to characterize the role of NK/NKT cells in murine experimental peritonitis. By injection with 200 μg αNK1.1 mAb (Hybridom PK136, ATCC) in 8 – 10 weeks old C57BL/6 mice 24 h prior to sepsis NK1.1+ cells (NK, NKT) were depleted. Sepsis was established by surgical insertion of a stent (size 18 gauge) in the ascending colon of experimental mice (CASP operation). Survival analysis and histochemistry were performed. αNK1.1 depleted animals revealed markedly increased susceptibility in the CASP model than controls (lethality 60% vs. 20% in controls) indicating a protective function of NK1.1+ cells in this model. Immunohistochemical analysis revealed a significant reduction of granulocytes and macrophages in livers from NK1.1 depleted animals 6 hand 12 h after surgery as compared to mice treated with control antibody. In conclusion, NK1.1+ cells play a crucial protective role in murine experimental peritonitis strongly suggesting an analogous function in human abdominal sepsis. Whether IFNγ mediates these effects remains to be elucidated.