Tom Erik Ruud

Impact of hypertonic saline on the release of selected cytokines after stimulation with LPS or peptidoglycan in ex vivo whole blood from healthy humans.

The question of specific immunomodulating qualities of hypertonic saline (HTS) has not been settled. It has proven difficult to distinguish between immunomodulation directly attributable to HTS and influence because of favorable circulatory effects. The nature of immune activator may also play a role. In a whole-blood model, we have investigated these relations further, with special emphasize on osmolalities usually found after recommended dosing. Blood from 10 healthy donors was exposed to osmolalities ranging from 295 to 480 mOsm/kg and stimulated with the two clinically relevant stimulators peptidoglycan (1 &mgr;g/mL) or LPS (10 ng/mL) for 6 h at 37°C. Leukocyte response was evaluated by measuring selected cytokines in the supernatant. Moderate hyperosmolality alone boosted the release of CXCL8/IL-8. The peptidoglycan-stimulated synthesis of pivotal proinflammatory cytokines was inhibited in an osmolality-dependent way, but statistically significant only at osmolalities above those attained after routine use of HTS, i.e., 310 mOsm/kg or greater: IL-6 (P < 0.05 at 315 mOsm/kg), IL-1&bgr;, and TNF-&agr; (P < 0.05 at 335 mOsm/kg). Similar effects were seen for the chemokine CCL3 and the anti-inflammatory cytokine IL-10. In contrast, the effects in cells stimulated with LPS were either lower or absent. Thus, osmolalities usually found after clinical use of HTS only modestly influenced the selected immune parameters, regardless of stimulator.

Systemic Administration of Enamel Matrix Derivative to Lipopolysaccharide-Challenged Pigs: Effects on the Inflammatory Response

BACKGROUNDnPeriodontitis is the primary clinical indication for enamel matrix derivative (EMD). Recent investigations, showing that EMD inhibits the production of tumor necrosis factor-alpha (TNF-alpha) when added to human whole blood, indicate a novel role for EMD as a modulator of systemic inflammation. In the present study, we investigated the systemic effects of EMD in lipopolysaccharide (LPS)-challenged pigs.nnnMETHODSnIn a preparatory study, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg/min). Thirty minutes later, a continuous infusion of LPS (1.7 mcg/kg/h) was started. An additional 12 pigs were randomized into two groups. Six of these animals were given the same treatment, except that EMD was administered 30 min after LPS. The remainder served as controls. The groups were compared according to organ injury and function, hemodynamics, and systemic markers of inflammation.nnnRESULTSnProphylactic administration of EMD triggered transient hemodynamic instability in two of seven pigs. In the randomized pigs, no or only nonspecific changes were observed in biopsies from vital organs, independent of treatment. Enamel matrix derivative did not modify systemic TNF-alpha, interleukin (IL)-1 beta, or IL-6 concentrations.nnnCONCLUSIONSnIn the formulation and dosages used, EMD did not modulate the inflammatory response. No true allergic or immunotoxic reactions were seen. To be usable for systemic application, a new formulation should be developed, or the active part of the protein(s) should be identified and produced in a soluble form designed for infusion. The potential of EMD as a systemic immune modulator is still unsettled.

An experimental study on effects of monopolar diathermy on the bile ducts

OBJECTIVEnTo study the effects of monopolar diathermy on the bile ducts in pigs.nnnDESIGNnExperimental study.nnnSETTINGnUniversity hospital, Norway.nnnMATERIALn18 pigs.nnnINTERVENTIONSnLaparotomy, application of diathermy at standard sites along the cystic duct, the bile ducts, and a cystic duct containing a metal clip, 3 to 12 times of 5 seconds duration at each site. Temperature was subsequently recorded at standard measurement points on the bile ducts. Twelve pigs were killed after three weeks for assessment of the bile ducts at necropsy.nnnMAIN OUTCOME MEASUREMENTSnIncrease in temperature in the bile duct walls and late changes in the bile ducts.nnnRESULTSnTemperature increased by 4-6 degrees C during 6 of 330 diathermy applications along the cystic duct, by 4-18 degrees C in 8 of 126 applications along the common bile duct, and by 4-11 degrees C at the clip in 9 of 54 applications. There were no macroscopic or microscopic changes in the bile ducts.nnnCONCLUSIONnMonopolar diathermy induced unexpected distant increases in the temperature of the bile duct walls and at a clip on the cystic duct probably because diathermy current energy was distributed along channels of high current conductivity.

In vivo characterization of ischemic small intestine using bioimpedance measurements.

The standard clinical method for the assessment of viability in ischemic small intestine is still visual inspection and palpation. This method is non-specific and unreliable, and requires a high level of clinical experience. Consequently, viable tissue might be removed, or irreversibly damaged tissue might be left in the body, which may both slow down patient recovery. Impedance spectroscopy has been used to measure changes in electrical parameters during ischemia in various tissues. The physical changes in the tissue at the cellular and structural levels after the onset of ischemia lead to time-variant changes in the electrical properties. We aimed to investigate the use of bioimpedance measurement to assess if the tissue is ischemic, and to assess the ischemic time duration. Measurements were performed on pigs (n = 7) using a novel two-electrode setup, with a Solartron 1260/1294 impedance gain-phase analyser. After induction of anaesthesia, an ischemic model with warm, full mesenteric arterial and venous occlusion on 30 cm of the jejunum was implemented. Electrodes were placed on the serosal surface of the ischemic jejunum, applying a constant voltage, and measuring the resulting electrical admittance. As a control, measurements were done on a fully perfused part of the jejunum in the same porcine model. The changes in tan δ (dielectric parameter), measured within a 6 h period of warm, full mesenteric occlusion ischemia in seven pigs, correlates with the onset and duration of ischemia. Tan δ measured in the ischemic part of the jejunum differed significantly from the control tissue, allowing us to determine if the tissue was ischemic or not (P < 0.0001, F = (1,75.13) 188.19). We also found that we could use tan δ to predict ischemic duration. This opens up the possibility of real-time monitoring and assessment of the presence and duration of small intestinal ischemia.

Effects of Infliximab and Hydrocortisone on In Vitro Cytokine Responses after Stimulation with Lipopolysaccharide

BACKGROUNDnBoth glucocorticosteroids and biologic drugs such as the tumor necrosis factor (TNF)-α antagonist infliximab are used often in the treatment of rheumatoid arthritis or inflammatory bowel disease. In severe disease, or if allergic reactions occur during treatment with infliximab, combined therapy with these drugs often is instituted. Combining infliximab and glucocorticosteroids may increase substantially the risk of severe opportunistic infections or dissemination of malignant tumors because of their additive effects as immunosuppressants.nnnMETHODSnIn a whole-blood in vitro model, we studied the influence of different doses of infliximab and hydrocortisone, either separately or in combination, on the synthesis of selected cytokines after stimulation with lipopolysaccharide (LPS).nnnRESULTSnHydrocortisone in therapeutic serum concentrations significantly inhibited the expression of a majority of the cytokines tested. Infliximab, in serum concentrations relevant to clinical situations, inhibited TNF-α activity significantly. This effect was potentiated when infliximab was combined with hydrocortisone. Similar effects were found using a low dose of infliximab combined with hydrocortisone. Infliximab alone inhibited the expression of the cytokines interleukin (IL)-1 receptor antagonist, monocyte chemoattractant protein-1, IL-8, and IL-12. Hydrocortisone in combination with low-dose infliximab potentiated the suppressive effects on TNF-α, IL-1β, IL-8, and macrophage inflammatory protein-1α synthesis.nnnCONCLUSIONSnImmune-modulating effects of infliximab were found both in clinically relevant doses and, most notably, in low doses reflecting serum concentrations found commonly in patients several months after the last injection. Infliximab potentiates the suppressive effects of hydrocortisone on cytokine synthesis.

Ischemia/reperfusion injury in porcine intestine - Viability assessment

AIM To investigate viability assessment of segmental small bowel ischemia/reperfusion in a porcine model. METHODS In 15 pigs, five or six 30-cm segments of jejunum were simultaneously made ischemic by clamping the mesenteric arteries and veins for 1 to 16 h. Reperfusion was initiated after different intervals of ischemia (1-8 h) and subsequently monitored for 5-15 h. The intestinal segments were regularly photographed and assessed visually and by palpation. Intraluminal lactate and glycerol concentrations were measured by microdialysis, and samples were collected for light microscopy and transmission electron microscopy. The histological changes were described and graded. RESULTS Using light microscopy, the jejunum was considered as viable until 6 h of ischemia, while with transmission electron microscopy the ischemic muscularis propria was considered viable until 5 h of ischemia. However, following ≥ 1 h of reperfusion, only segments that had been ischemic for ≤ 3 h appeared viable, suggesting a possible upper limit for viability in the porcine mesenteric occlusion model. Although intraluminal microdialysis allowed us to closely monitor the onset and duration of ischemia and the onset of reperfusion, we were unable to find sufficient level of association between tissue viability and metabolic markers to conclude that microdialysis is clinically relevant for viability assessment. Evaluation of color and motility appears to be poor indicators of intestinal viability. CONCLUSION Three hours of total ischemia of the small bowel followed by reperfusion appears to be the upper limit for viability in this porcine mesenteric ischemia model.

Regulation of CCN1 (Cyr61) in a porcine model of intestinal ischemia/reperfusion:

Intestinal ischemia is a serious condition that may lead to both local and systemic inflammatory responses. Restoration of blood supply (reperfusion) to ischemic tissues often increases the extent of the tissue injury. Cysteine-rich angiogenic inducer 61 (Cyr61)/CCN1 is an extracellular matrix-associated signaling protein that has diverse functions. CCN1 is highly expressed at sites of inflammation and wound repair, and may modify cell responses. This study aimed to investigate regulation and cellular distribution of CCN1 in intestinal ischemia/reperfusion (I/R) injury in pigs. After intestinal I/R, increased expression of CCN1 was detected by quantitative RT-PCR, Western blot analysis and immunohistochemistry compared with non-ischemic intestine. Immunoflorescence staining revealed that CCN1 was mainly up-regulated in intestinal mucosa after intestinal I/R. Microvillus epithelial cells and vascular endothelial cells were strongly positive for CCN1 in intestinal I/R, while natural killer cells and/or subsets of neutrophils were only modestly positive for CCN1. Furthermore, blood samples taken from the portal and caval veins during ischemia and after reperfusion showed no change of the CCN1 levels, indicating that CCN1 was locally regulated. In conclusion, these observations show, for the first time, that the CCN1 molecule is up-regulated in response to intestinal I/R in a local manner.

Small intestinal ischemia and reperfusion—bioimpedance measurements

OBJECTIVEnTrans-intestinal bioimpedance measurements have previously been used to investigate changes in electrical parameters during 6u2009h of ischemia in the small intestine. Knowledge is lacking regarding the time course of trans-intestinal bioimpedance parameters during reperfusion. As reperfusion is an important part in the clinical treatment of intestinal ischemia, we need to know how it affects the bioimpedance measurements.nnnAPPROACHnWe performed bioimpedance measurements, using a two-electrode setup on selected segments of the jejunum in 15 pigs. A controlled voltage signal was applied while measuring the resulting current. In each pig, five or six 30u2009cm segments of the jejunum were made ischemic by clamping the mesenteric arteries and veins creating segments with ischemia from 1-16u2009h duration. Reperfusion was initiated at selected time intervals of ischemia, and measured for 5-15u2009h afterwards.nnnMAIN RESULTSnThe tan δ parameter (loss tangent) was different (pu2009u2009<u2009u20090.016) comparing ischemic and control tissue for the duration of the experiment (16u2009h). Comparing the control tissue 30u2009cm from the ischemic area with the control tissue 60u2009cm from the ischemic tissue, we found that the mean tan δ amplitude in the frequency range (3900-6300 Hz) was significantly higher (pu2009u2009<u2009u20090.036) in the proximal control after 10u2009h of experiment duration. After reperfusion, the time development of tanδm (loss tangent maximum over a frequency range) amplitude and frequency overlapped and periodically increased above the tanδm in the ischemic intestine. Dependent on the ischemic duration pre-reperfusion, the initial increase in tan δ stabilizes or increases drastically over time, compared to the tan δ amplitude of the ischemic tissue.nnnSIGNIFICANCEnAs during ischemia, the electrical parameters during reperfusion also follow a characteristic time-course, depending on the ischemic exposure before pre-reperfusion. The temporal changes in electrical parameters during small intestinal ischemia followed by reperfusion provides important information for assessment of tissue injury.

Ischemic small intestine—in vivo versus ex vivo bioimpedance measurements

OBJECTIVEnBioimpedance has been used to investigate changes in electrical parameters during ischemia in various tissues. The small intestine is a multi-layered structure, with several distinct tissue types, and ischemia related changes occur at different times in the different intestinal layers. When investigating how the electrical properties in the small intestine is affected by ischemia, some researchers have used ex vivo models while others have used in vivo models. In this study, we compare ischemic time development of electrical parameters in ischemic in vivo versus ex vivo small intestine.nnnAPPROACHnMeasurements were performed using a two-electrode setup, with a Solartron 1260/1294 impedance gain-phase analyser. Electrodes were placed on the surface of ischemic pig jejunum, applying a voltage and measuring the resulting electrical admittance. In each pig, 4 segments of the jejunum were made ischemic by clamping the mesenteric arteries and veins, resulting in a 30u2009cm central zone of warm ischemia and edema. The in vivo part of the experiment lasted 10u2009h, after which 3 pieces of perfused small intestine were resected, stored in Ringer-acetat at 38 °C, and measured during a 10u2009h ex vivo experiment. Main results and significance: We found significant differences (pu2009u2009<u2009u20090.0001) between the values of electric parameters when comparing the in vivo and ex vivo measurements as a function of ischemic time development. We also observed some similarities in the trends. In vivo, we measured an overall decrease in impedance during the duration of the experiment, probably as a result from the formation of edema. Ex vivo, the low frequency impedance increased initially for approximately 3u2009h before starting to decrease.

SYSTEMIC PETIDOGLYCAN (PEPG) STIMULATES PROINFLAMMATORY RESPONSES IN SYNERGY WITH LIPOPOLYSACCHARIDE (LPS) IN AN IN VIVO AND IN VITRO PORCINE MODEL

TOWARDS RESOLVING THE CHALLENGE OF SEPSIS DIAGNOSTIC. Thomas Herget* and Thomas Joos . *Merck KGaA, Darmstadt, Germany; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany Biomarkers have proven to be very useful in clinical conditions such as heart attack, stroke and cancer. There are characteristics linked to sepsis like in blood pressure, body temperature and heart rate. Efforts over the last decade to improve diagnosis for infectious inflammation have been unsuccessful in identifying a single and universal biomarker that provides sufficiently high sensitivity and specificity. In gramnegative septicemia and following major abdominal trauma, the determination of endotoxin continues to be a leading candidate which could become adopted into clinical practice. The importance of endotoxin measurement continues to grow as more clinicians recognize the added value of measuring endotoxin in critically ill patients and with the emergence of major pharmaceutical trials directly targeting endotoxin in the bloodstream. However, hundreds of other candidates potentially serving as biomarker for sepsis have been recently described, e.g. cysteinyl-leukotriene (LTC4) generation, procalcitonin (PCT) and C-reactive protein (CRP). However, none of them fulfils the criteria requested by clinicians, namely being specific and sensitive. The presentation will discuss criteria for a sepsis biomarker, will give an overview of obtaining samples from appropriate cell systems and from patients. Furthermore, tools will be described to identify marker candidates on genetic-, proteinand metabolite level. The integration of these data sets covering e.g. signal transduction, protein : protein interaction, gene expression with the help of bioinformatics and systems biology will help to validate such candidates. The final goal is manufacturing a robust diagnostic device for clinical routine work. A solid sepsis diagnostics method will be beneficial for patients, but also for the healthcare systems and will open challenges for the pharmaceutical industry.