Thomas Volk

Immunomodulatory therapies in sepsis

Abstract Despite advances in critical care medicine, mortality from sepsis in ICU patients remains high. In response to several infectious and non-infectious stimuli, monocytes/macrophages release a number of mediators, including cytokines, involved in the proinflammatory response that underlies sepsis. The excessive release of these mediators results in the development of whole body inflammation, and plays an important role in the pathogenesis of sepsis and septic shock. In addition, patients with sepsis also undergo an anti-inflammatory phase (the compensatory anti-inflammatory response syndrome) and at times, a mixed response with both pro-and anti-inflammatory components (the mixed antagonistic response syndrome). The initial systemic hyperinflammation is caused by production of inflammatory cytokines, especially tumour necrosis factor-α (TNF-α), and also interleukin-1 (IL-1), IL-6, and interferon gamma, which act synergistically with TNF-α in inducing shock in animal models. However, clinical trials aimed at downregulating these mediators using antibodies against endotoxin, TNF-α, antagonists of IL-1 or platelet activating factor have proved to be uniformly disappointing. Not only have these agents been found to have no effect, but they may also increase mortality. One of the reasons for such failure may be the lack of precise immunological monitoring during the course of sepsis.¶We have recently demonstrated that sepsis shows a biphasic immunological pattern during the initial and later phase: the early hyperinflammatory phase is counterbalanced by an anti-inflammatory response which may lead to a hypoinflammatory state. The latter is associated with immunodeficiency that is characterised by monocytic deactivation, so-called immunoparalysis. Interferon gamma-1 b has an immunoregulatory effect in patients with immunoparalysis during the compensatory anti-inflammatory response syndrome, not only restoring levels of HLA-DR expression but also re-establishing the ability of monocytes to secrete cytokines such as TNF-α. By monitoring immune status in septic patients, targeted intervention may lead to more success in immunomodulation of sepsis.

Hyperprocalcitonemia in patients with noninfectious SIRS and pulmonary dysfunction associated with cardiopulmonary bypass

Background The incidence of noninfectious systemic inflammatory response syndrome (SIRS) associated with coronary artery bypass surgery and the potential role of several inflammatory parameters as early markers of pulmonary dysfunction induced by cardiopulmonary bypass (CPB) were investigated. Methods Forty patients undergoing elective coronary artery bypass surgery were studied prospectively. Perioperative lung function was monitored using the lung injury score introduced by Murray and colleagues, by measuring venous admixture (Qs/Qt), and, in some cases, by measuring extravascular lung water. Serum concentrations of the inflammatory parameters (procalcitonin, interleukin‐6, sL‐selectin, leukocyte elastase, neopterin, leukocyte counts, and C‐reactive protein) were determined sequentially. The American College of Chest Physicians‐Society of Critical Care Medicine classification system was used to diagnose SIRS. Results According to the entry criteria, SIRS developed in 17 (42%) patients after operation. Nine patients of this group showed signs of acute pulmonary impairment, whereas patients without SIRS had no lung injury. In all patients with acute lung injury, distinct increases in procalcitonin concentrations ranging from 5.1 to 14.3 ng/ml were measured. In patients with SIRS but without acute lung injury and in patients without SIRS, none or only negligible increases in serum concentrations of procalcitonin were seen. Compared with procalcitonin, other inflammatory parameters investigated were less sensitive and less specific to indicate pulmonary dysfunction secondary to CPB. Conclusions Procalcitonin seems to be an appropriate parameter indicating the early development of severe noninfectious SIRS and for predicting pulmonary dysfunction secondary to CPB.

Oxidized proteins as a marker of oxidative stress during coronary heart surgery

The measurement of the degree of oxidative stress in patients often causes problems because of the lack of useful parameters. Therefore, we used an ELISA technique to evaluate serum protein carbonyls as a parameter of oxidative stress in patients during coronary heart surgery. Protein carbonyls were detected in serum samples of 14 patients undergoing coronary surgery and cardiopulmonary artery bypass grafting. A clear 2- to 3-fold increase in protein carbonyls in serum samples taken from human venous coronary sinus could be detected in the reperfusion period of the heart. We compared these data with markers of oxidative stress previously used, such as the glutathione status and the lipid peroxidation product malondialdehyde (MDA). Strong correlations of the protein carbonyl formation with MDA (r2 = 0.86) and oxidized glutathione (r2 = 0.81) were found in the early reperfusion stage. Increased levels of oxidized glutathione and MDA were detected only in the early reperfusion period. In contrast, the serum protein carbonyl content remained elevated for several hours, indicating a considerably slower serum clearance of oxidized proteins compared with that of lipid peroxidation products and the normalization of the glutathione status. We therefore concluded that the measurement of serum carbonyls by this ELISA technique is suitable to detect oxidative stress in serum samples of patients. The relative stability of the parameter makes the protein carbonyl detection even more valuable for clinical purposes.

Postoperative Epidural Anesthesia Preserves Lymphocyte, but Not Monocyte, Immune Function After Major Spine Surgery

Extensive spine surgery is associated with postsurgical pain. Epidural pain therapy may reduce postoperative stress responses and thereby influence immune functions. In a randomized, controlled, double-blinded prospective trial, 54 patients received either conventional patient-controlled IV analgesia (PCIA; morphine 3 mg/15 min) or patient-controlled epidural analgesia (PCEA; 0.125% ropivacaine plus sufentanil 1 &mgr;g/mL at a base rate of 12 mL/h and bolus application of 5 mL/15 min). Circulating cytokines, C-reactive protein (CRP), cortisol, and cell-surface receptor expression of immune cells (cluster of differentiation [CD]14, human leukocyte antigen-DR, CD86, CD71, CD3, CD4, CD8, CD16, and CD19) were measured perioperatively to characterize immunological functions. PCEA, compared with PCIA, had no influence on altered levels of circulating cytokines (interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor-&agr;, monocyte chemoattractant protein-1, and macrophage inhibitory factor) or indicators of the stress response (CRP and cortisol). Also, no significant difference was found in monocyte numbers or their human leukocyte antigen-DR, CD86, or CD71 expression. In contrast, the postoperative decrease in B lymphocytes and T-helper cells was significant in the PCEA group. Natural killer cells decreased significantly in patients receiving PCEA compared with PCIA. Therefore, postoperative epidural pain therapy has no influence on monocyte functions but reduces natural killer cells and preserves B-cell and T-helper cell populations. Epidural analgesia thus influences the specific rather than the innate immune system and potentially blunts the postsurgical lymphocyte depression, which is relevant for infectious resistance.

A Comparison of Seal in Seven Supraglottic Airway Devices Using a Cadaver Model of Elevated Esophageal Pressure

BACKGROUND:Supraglottic airway devices are increasingly important in clinical anesthesia and prehospital emergency medicine, but there are only few data to assess the risk for aspiration. We designed this study to compare the seal of seven supraglottic airway devices in a cadaver model of elevated esophageal pressure. METHODS:The classic laryngeal mask airway, laryngeal mask airway ProSeal™, intubating laryngeal mask airway Fastrach™, laryngeal tube™, laryngeal tube LTS II™, Combitube™, and Easytube™ were inserted into unfixed human cadavers with an exposed esophagus that had been connected to a water column of 130 cm height. Slow and fast increases of esophageal pressure were performed and the water pressure at which leakage appeared was registered. RESULTS:The Combitube, Easytube, and intubating laryngeal mask Fastrach withstood the water pressure up to more than 120 cm H2O. The laryngeal mask airway ProSeal, laryngeal tube, and laryngeal tube LTS II were able to block the esophagus until 72–82 cm H2O. The classic laryngeal mask airway showed leakage at 48 cm H2O, but only minor leakage was found in the trachea. Devices with an additional esophageal drain tube drained fluid sufficiently without pulmonary aspiration. CONCLUSIONS:Concerning the risk of aspiration, the use of devices with an additional esophageal drainage lumen might be superior for use in patients with an increased risk of aspiration. The Combitube, Easytube, and intubating laryngeal mask Fastrach showed the best capacity to withstand an increase of esophageal pressure.

Postoperative analgesia after major spine surgery: patient-controlled epidural analgesia versus patient-controlled intravenous analgesia.

BACKGROUND: Spinal fusion surgery causes severe postoperative pain, hampering reconvalescense. We investigated the efficacy of patient-controlled epidural analgesia (PCEA) in a prospective, double-blind, randomized, controlled comparison with patient-controlled IV analgesia (PCIA). METHODS: After lumbar anterior-posterior fusion receiving an epidural catheter intraoperatively, 72 patients were given either PCEA (ropivacaine 0.125% and sufentanil 1.0 &mgr;g/mL at 14 mL/h; bolus: 5 mL; lockout time: 15 min) and IV placebo or PCIA (morphine 2.0 mg/mL; bolus: 3 mg; lockout time: 15 min) and epidural placebo. Pain levels (visual analog scale 0-10), functional capabilities (turning in bed, standing, and walking), analgesic consumption, and side effects were evaluated until 72 h after surgery. RESULTS: Fourteen patients were excluded by predetermined criteria, leaving 58 patients for data analysis. Pain levels at rest and during mobilization were significantly lower in the PCEA when compared with that in the PCIA group throughout the study period (P < 0.0001 in all cases). Time until able to turn in bed was achieved earlier in the PCEA group (P < 0.05). Patients in the PCEA group were significantly more satisfied with pain therapy (P < 0.01). CONCLUSION: We conclude that PCEA with ropivacaine and sufentanil, using intraoperatively placed epidural catheters, provides superior analgesia and higher patient satisfaction when compared with PCIA after spinal fusion surgery.

Secretion of MCP-1 and IL-6 by cytokine stimulated production of reactive oxygen species in endothelial cells.

Endothelial cells are known to produce reactive oxygen species by several mechanisms. Functional consequences of increased production of reactive oxygen species were investigated in vitro after stimulation with several proinflammatory cytokines. Time dependent increases in DCF-fluorescence as a measure of reactive oxygen load were quantified in single cells after incubation with TNF-α, IL-1 and IFN-γ. The increased DCF-fluorescence was inhibited by cell permeant antioxidative substances Tiron and Tempol. NMMA, an inhibitor of nitric oxide synthase reduced endothelial DCF-fluorescence only marginally, indicating a minor participation of nitric oxide production in this detection system. Cytokine induced endothelial DCF-fluorescence increased in the presence of NADH, whereas coincubation with NADPH or xanthine was without effect. Flavoenzyme inhibitor diphenyliodonium abolished stimulated DCF-fluorescence. Cytokine induced release of MCP-1 and IL-6 by endothelial cells was completely inhibited in the presence of Tiron and Tempol, whereas NMMA was less effective. Collectively these data indicate that cytokine stimulated endothelial cells increase their reactive oxygen species production probably via NADH oxidase and this production may critically be involved in the secretion of MCP-1 and IL-6.

Influence of aminosteroid and glucocorticoid treatment on inflammation and immune function during cardiopulmonary bypass

Objective During cardiopulmonary bypass, inflammation and immunosuppression is present. We measured circulating mediators and monocyte-based functions and tested the hypothesis that these variables are influenced by methylprednisolone (MP) or tirilazad mesylate (TM) treatment. Design Randomized, controlled, double-blind prospective trial. Setting A university hospital. Patients Thirty-nine patients scheduled for conventional coronary surgery with three-vessel disease. Interventions Preoperative application of MP (15 mg/kg) or TM (10 mg/kg) compared with placebo (PL). Measurements and Main Results Circulating proinflammatory markers including interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1, and C-reactive protein were all decreased by MP treatment but not by TM treatment. Whereas rapid increases in circulating anti-inflammatory IL-10 were superinduced by MP but not TM, plasma levels of IL-1RA and transforming growth factor &bgr; were not altered by either treatment. Decreased ex vivo lipopolysaccharide-stimulated secretion of tumor necrosis factor &agr; was prolonged after MP treatment but not after TM treatment. Perioperative stimulated secretion of IL-12 and interferon &ggr; was diminished in all groups, whereas ex vivo IL-1RA secretion tended to increase in all groups. Depression of monocyte surface expression of HLA-DR was significantly greater in patients treated with MP, whereas CD14 expression did not change. Conclusions These data confirm that, during cardiopulmonary bypass, pro- and anti-inflammatory systems are activated at the same time, whereas monocyte-based immune functions are depressed. Treatment with MP abrogates proinflammatory mediators and induces a shift toward anti-inflammation at the cost of further functional monocyte deficits, whereas treatment with TM apparently has neither anti-inflammatory nor immunosuppressive actions in this setting.

Ferritin Oxidation in Vitro: Implication of Iron Release and Degradation by the 20S Proteasome

Ferritin, the major iron storage protein in mammalian cells, was treated with various concentrations of different oxidants: xanthine/xanthine oxidase, Sin‐1 (3‐morpholinosydnonimine, purchased from Alexis, Grunberg, Germany), DEA‐NO (Diethylamine NONOate, purchased from Calblochem‐Novabiochem, Schwalbach, Germany), and hydrogen peroxide. The proteolytic susceptibility towards the isolated 20S proteasome of untreated ferritin and oxidized ferritin was measured in parallel with the iron liberated by these oxidants. With increasing hydrogen peroxide, Sin‐1, and xanthine oxidase concentrations, the measured proteasomal degradation of ferritin also increased. At higher oxidant concentrations, however, the proteolytic susceptibility began to decrease. The oxidation of ferritin by DEA‐NO was accompanied by a lesser increase of proteolytic susceptibility in comparison with the effects of the other oxidants. Addition of DEA‐NO to Sin‐1 suppressed the increase in proteolytic susceptibility of ferritin, whereas adding xanthine/xanthine oxidase had no additional effect. Iron was liberated readily from ferritin as a result of the oxidation process, although the increase in proteolytic susceptibility was not always correlated to the iron release. In fact, the degradation of oxidatively damaged ferritin was not accompanied by a further increase of free iron. Therefore, we conclude that the proteasome is a secondary antioxidative defense system that degrades only nonfunctional ferritin.

Nitric oxide induces transient Ca2+ changes in endothelial cells independent of cGMP

Ca2+ changes induced by nitric oxide (NO·) were investigated in cultured human endothelial cells. Sodium nitroprusside (SNP) (1–100 μmol/L) and S‐Nitroso‐N‐acetylpenicillamine (SNAP) (100 μmol/L) were used as NO· donors. The cytoplasmatic Ca2+ concentration was calculated using ratiometric FURA2 fluorescence measurements. Both NO· donors caused transient oscillatory Ca2+ changes, which were not detectable in the presence of oxyhemoglobin (50 μmol/L). Digital ratio imaging revealed initiation sites within cells where Ca2+ increases started spreading, which indicates that nonuniformly distributed targets might be involved in these reactions. Calcium was released from intracellular stores as indicated by experiments performed in Ca2+‐free buffer. L‐type Ca2+‐channel blocker diltiazem (100 μmol/L) was not able to block these responses. NO·‐induced Ca2+ release from intracellular stores caused capacitative Ca2+ entry. Both thapsigargin (1 μmol/L) and cyclopiazonic acid (10 μmol/L) inhibited the SNP response completely, whereas neither ryanodine (up to 100 μmol/L) nor dantrolene (100 μmol/L) was able to inhibit Ca2+ changes induced by SNP, indicating that primarily inositol 1,4,5‐triphosphate (IP3)‐dependent stores are released upon stimulation with NO·. A small inhibitory effect of ATP‐ and SNP‐induced peak [Ca2+]i increase was measured in the presence of both caffeine (20 mmol/L) and procaine (1 mmol/L). Evidence is presented that cGMP is not involved in NO·‐induced Ca2+ signals, as neither inhibitors of guanylate cyclase (methylene blue and LY (83583) nor cell permeant analogues of cGMP altered or simulated [Ca2+]i changes. An inhibitor of cGMP‐dependent protein kinase was also ineffective. We therefore propose that endothelial cells have specific targets proximal or at IP3 receptors to induce Ca2+ changes in endothelial cells stimulated with NO·. J. Cell. Physiol. 172:296–305, 1997.