LPS does not induce changes in hepatocellular microtubule cytoskeleton

Abstract

Critical Care 1998, 2(Suppl 1):P001 Background: Whole body hyperthermia induced by radiative systems has been used in therapy of malignant diseases for more than ten years. Von Ardenne and co-workers have developed the 'systemiche Krebs-Mehrschritt-Therapic' (sKMT), a combined regime including whole body hyperthermia of 42°C, induced hyperglycaemia and relative hyperoxaemia with additional application of chemotherapy. This concept has been employed in a phase I/II clinical study for patients with metastatic colorectal carcinoma at the Virchow-Klinikum since January 1997. Methods: The sKMT concept was performed eleven times under intravenous general anaesthesia, avoiding volatile anaesthetics. Core temperatures of up to 42°C were reached stepwise by warming with infrared-A-radiation (IRATHERM 2000®). During the whole procedure blood glucose levels of 380-450 mg/dl were maintained as well as PaO 2 levels above 200 mmHg. Extensive invasive monitoring was performed in all patients including measurements with the REF-Ox-Pulmonary artery catheter with continuous measuring of mixed venous saturation (Baxter Explorer®) and invasive monitoring of arterial blood pressure. Data for calculation of hemodynamic and gas exchange parameters were collected four times, at temperatures of 37°C, 40°C, 41.8-42°C and 39°C, during measurements FiO 2 was 1.0 at all times. Fluids were given in order to keep central-venous and Wedge pressure within normal range during the whole procedure. Statistics were performed using the Wilcoxon Test. Results: Statistically significant differences were found between heart rate, cardiac index and systemic vascular resistance comparing data at 37°C and 42°C. Heart rate and cardiac index increased to a maximum at 42°C (P < 0.0001) whereas systemic vascular resistance had its minimum at 42°C (P < 0.0001). Mean arterial pressure dropped with increasing temperature, differences were not significant. Calculation of stroke volume index and ventricular volumes showed only a slight decrease in endsystolic volumes with increasing temperature, the resulting differences in right ventricular ejection fraction were marginally significant (P = 0.038) comparing 42°C to baseline. Right ventricular stroke work index as well as mean pulmonary arterial pressure increased at 42°C (P = 0.0115 and P = 0.0037), pulmonary vascular resistance only dropped little compared to systemic vascular resistance, left ventricular stroke work index even dropped with increasing temperature, though showing no significant difference. Values for mixed venous oxygen saturation did not vary during therapy, pulmonary right-left shunt showed a temperature associated increase (P = 0.0323) to a maximum at 42°C. Conclusion: Under the procedure of sKMT cardiac function in patients, who do not have any pre-existing cardiac impairment, can be maintained almost unchanged, ie with normal right and left ventricular pressure, despite an increase in right ventricular stroke work Acknowledegment: Supported by Deutsche Krebshilfe. P002 Induced hyperthermia causes significant changes in lymphocytes Critical Care 1998, 2(Suppl 1):P002 Background: Changes in lymphocyte subpopulations are determined under several clinical conditions eg during activation of the immune system. Our aim was to analyze the influence of induced elevated body temperatures on lymphocytes in patients without infections or other physiological stimulators of the immune system. Therefore we examined blood of patients with metastatic colorectal carcinoma during whole body hyperthermia of 42°C caused by infrared-A-radiation. This is used as part of so called 'systemische Krebs-Mehrschritt-Therapie' (sKMT), which was started as a phase I/II clinical study at Virchow-Klinikum in 1997. Methods: Lymphocyte subpopulations were investigated by flow-cytometry-analysis. Blood samples were obtained before beginning of therapy at 37°C, at 40°C, at the end of the plateau of 42°C and after therapy at 37°C again. Time between investigations was about 2 h. Subpopulations were natural killer cells, T-Cells, IL2-Receptor on T-Cells, T4-Cells and T8-Cells. Cell counts were compared by using a Wilcoxon rank sum test. Results: The number of lymphocytes per nl decreased significantly from 37°C to 42°C (Fig. 1). This effect was mainly caused by a significant decrease of the absolute T4-Cell count and a slight decrease of the T8-Cell count with a resulting significant decrease of T-Cells. In addition, IL2-Receptor expression on T-Cells, as a marker for activation, decreased significantly. In contrast, the number of natural killer cells per nl Critical Care 1998, Volume 2 Suppl 1 http://ccforum.com/supplements/2/S1 increased. Looking for changes in relation between lymphocyte subpopulations, we found a significant percentual decrease of T4-Cells (Fig. 2), no percentual changes in T8-Cells but a significant percentual increase of natural killer cells (Fig. 3). Effects were reversible and at the last time-point at 37°C all examined parameters showed a tendency to the initial values. Conclusions: Elevated body temperatures up to 42°C induce a change in lymphocytes which is similar to early responses of the immune system to other stress situations or host response. For example natural killer cells are known to increase in the early phase after severe trauma, whereas the number of T4-Cells decreases in these patients. Thus, isolated induced hyperthermia in absence of infections or other physiological stimulators of the immune system seems to cause a kind of host response. It seems remarkable, that these effects were reversible in a very short time-period after decrease of temperature. Acknowledgement: Supported by Deutsche Krebshilfe. P003 Relationship between reactive hemophagocytic syndrome (RHS) and multiple organ system failure (MOSF) F Gauvin, B Toledano, M David, J Lacroix Sainte-Justine Hospital, 3175 Côte Sainte-Catherine, Montréal (Québec), Canada H3T 1C5 Critical Care 1998, 2(Suppl 1):P003 Objective: To report two cases which show that severe RHS can be reversible, can be a cause of cytopenia in the ICU, and can be associated with MOSF. In both cases, a bone marrow aspirate showed histiocytes, hypocellularity of all cell lines, and hemo-and erythrophagocytosis. Case reports: 1) A 3 year-old boy with Mucha-Haberman syndrome was admitted to the PICU for septic shock (Staphylococcus epidermidis and Candida in blood), acute respiratory distress syndrome (ARDS), capillary leak, acute renal failure, liver dysfunction, MOSF and RHS. PRISM II score was 13. The pancytopenia worsened (WBC: 900 cells/mm 3 ; Hb: 59 g/l; Plt: 36000/mm 3), then resolved 2 months later. The patient required mechanical ventilation for 6 weeks. Length of stay in ICU was 2 months. 2) A previously healthy 4 year-old girl was admitted to the PICU for respiratory failure. PRISM II score was 23. She developed ARDS, cardiomyopathy with shock and complete atrio-ventricular block, capillary leak, liver dysfunction and RHS (WBC: 1900 cells/mm 3 ; Hb: 65 g/l; Plt: 58000/mm 3). Serology for respiratory syncitial virus was positive. The duration of RHS was 20 days; length of mechanical ventilation was 16 days, and length of stay in the PICU was 3 weeks. Conclusion: Both patients recovered completely; thus severe cases of pediatric RHS can be reversible. These cases also show that RHS may be a significant cause of cytopenia in the PICU. Data in the literature and these cases suggest that RHS may be an integral part of MOSF: 1) RHS appeared and disappeared with MOSF; 2) RHS and MOSF share similar trigger events, such as infection or neoplasia; 3) increased level of cytokines (IL-6, IFN-alpha and IL-2 receptor, etc.) are present both in MOSF and RHS [1-3]. Critical Care 1998, 2(Suppl 1):P004 Introduction: Lipopolysaccharides (LPS) are known to be involved in the pathogenesis of septic shock and multiorgan failure. It has been demonstrated that LPS may cause changes in monocyte cytoskeleton and directly influence assemblation of isolated microtubuli. As liver failure is increasingly observed during septic shock we estimated the influence of LPS on microtubule cytoskeleton of cultivated hepatocytes and human blood monocytes. Methods: HepG2 cells, murine hepatocytes, or human monocytes (positive control) were incubated with LPS FITC labelled or unlabelled (0.1-200 μg/ml) up to 48 h. After staining with antibodies to tubulin and tau proteins cells were analysed by fluorescence and laser scan confocal microscopy. Activation of MAP-kinases was investigated by Western Blot using phosphospecific antibodies. Results: Immunofluorescence revealed that the cytoskeleton of hepatocytes was not affected by LPS. Furthermore, no phosphorylation of MAP-kinases was found after LPS-incubation. Mouse hepatocytes and HepG2 cells did not accumulate FITC labelled LPS. In contrast, human blood monocytes showed an accumulation of FITC-LPS, an activation of MAP-kinases and changes in microtubule cytoskeleton. Conclusions: Our results might explain the frequently observed late onset of liver failure during sepsis-syndrom. Further studies on possible protective factors in hepatocytes and the complex cooperation between LPS and cytokines leading to hepatocellular damage are necessary. Acknowledgement: Supported by Deutsche Forschungsgemeinschaft Re 653/5-1 and Thuringian Ministry of Science, Research and Culture F 3.1-908/7-143. P005 HepG2 hepatocytes express IFN-g, TNF-a, TGF-b, M-CSF, oncostatin-M, ICAM-1, IL-4, IL-5, IL-7, IL-10, IL-11, IL-12 and IL-6 receptor genes in vitro