[Design and evaluation of the University of Munich courses on research trials]. / Konzept und Evaluierung der Münchner Prüfarztkurse.

INTRODUCTION: The growing importance of clinical research in Germany requires physicians who are adequately trained to conduct clinical trials. In November 2007, the Coordination Centre for Surgical Studies at the University of Munich organised its third course for clinical investigators. The course was evaluated by the participants. PARTICIPANTS AND PROGRAMME: Participants included 1st-year residents as well as specialists mostly from surgical departments; many already possessed significant experience with clinical trials. In our course, special emphasis was put on the practical aspects of conducting clinical trials. EVALUATION: On a scale from 1 to 6 (in German grading, 1 is the best score), participants rated content at 1.5, understanding at 1.6, and learning effect at 1.8. Careful preparation and a clearly structured curriculum resulted in significantly better evaluation of this course than of preceding courses (content P=0.015, understanding P=0.007, learning effect P=0.018). DISCUSSION: To realise projects of translational investigation and to participate in interdisciplinary clinical trials, surgeons in academic centres should obtain basic knowledge on clinical and pharmaceutical trials. Participants' statements on their own experience as clinical investigators underline the need for adequate training in this field. This course conveys essential knowledge of good medical practice and critical review of medical literature. We view this knowledge as a basic skill for modern medical practice and have therefore integrated the course into basic surgical training at our department. CONCLUSION: The concept of training for clinical investigation conducted at a surgical department is well established, and the participants' positive feedback motivates us to continue organising such courses.

Depressed T cell-derived IFN-gamma following trauma-hemorrhage: a potential mechanism for diminished APC responses.

INTRODUCTION: Prolonged immunosuppression has been demonstrated after trauma-hemorrhage resulting in an increased susceptibility to sepsis. The contribution of antigen-presenting cells (APC) vs T cells to this diminished immune response, however, remains unknown. MATERIALS AND METHODS: To study this, male mice were trauma-hemorrhaged (35 +/- 5 mmHg for 90 min and resuscitation) or sham operated. At 24 h thereafter, spleens were harvested and T cells (via Microbeads) and APC (via adherence) were isolated. Cocultures of combined T cells and APC were established for 48 h, stimulated with ConA and LPS. The T cell-derived cytokine IFN-gamma and IL-12 for APC responses were measured in the supernatants by the multiplex assay. RESULTS: The release of IFN-gamma was suppressed by T cells after trauma-hemorrhage irrespective of whether sham or trauma-hemorrhage APC were added. Trauma-hemorrhaged APC did not affect T cells-derived IFN-gamma release by sham T cells. In contrast, trauma-hemorrhaged T cells depressed the release of IL-12 by APC. The release of IL-12 by trauma-hemorrhaged APC was not altered when sham T cells were cocultured. CONCLUSION: Prolonged immunosuppression after trauma-hemorrhage appears to be predominantly due to diminished T cell function. Thus, attempts to prevent immunodysfunction should be directed towards T cells.

Immunomodulation during sepsis in organ transplanted children.

Newer immunosuppressive agents have dramatically reduced the rates of acute graft rejection over the last decade but may have exacerbated the problem of post-transplant infections. Causes of early mortality include graft dysfunction and sepsis. Late mortality occurs mainly due to sepsis. An excessive inflammatory response followed with a dramatic paralysis of cell-mediated immunity has been documented in septic patients. In transplanted individuals the pathophysiological changes of the immune response are further complicated by immunosuppressive agents. This article will focus on the effect of immunosuppressive agents and sepsis on cell-mediated immune responses. Moreover, potentially promising immunomodulatory approaches, i.e. human activated protein C, immunomodulatory diets containing L-arginine and fish oil, selective cytokine blockade, platelet-activating factor receptor antagonist, LPS receptor CD14 blockade and G-CSF, for the treatment of immunodysfunction in septic patients will be outlined in this review article. Most of them, however, have not been tested in the clinical arena in transplanted patients. Thus, the main part of the article, immunomodulation during sepsis in organ transplanted children is quite speculative and based on immunomodulatory strategies in other non-transplanted septic patients.

Differences in the expression of LPS-receptors are not responsible for the sex-specific immune response after trauma and hemorrhagic shock.

Several studies demonstrated a sex-specific cytokine secretion by macrophages following trauma-hemorrhage (T-H) and incubation with lipopolysaccharide A (LPS). Although LPS is known to act via the receptors CD14 and TLR4 on macrophages, it remains unknown whether differences in LPS receptor expression in males and females may be responsible for the gender-specific LPS induced cytokine response following (T-H). To study this, male and proestrus female mice (C3H/HeN) were subjected to trauma (laparotomy) followed by hemorrhage or sham operation. At 2 h thereafter, SMphi and PMphi were harvested and cultured for 2 h. The expression of CD14 and TLR4 was measured by flow cytometry on unstimulated SMphi and PMphi as well as after LPS stimulation. The results indicate that the expression of CD14 and TLR4 on SMphi and PMphi from female and male mice was similar in sham-operated animals and after (T-H). Incubation of macrophages with LPS did not alter CD14 and TLR4 expression in the study groups. Thus, the sex specific LPS induced cytokine secretion after (T-H) is not caused by differences in LPS receptor expression on Mphi of male and female mice.

Effects of perioperative recombinant human IFN-gamma (rHuIFN-gamma) application in vivo on T cell response.

In spite of the well-known immunoregulatory effects of recombinant human interferon-gamma (rHuIFN-gamma), in vitro clinical trials in trauma patients remain inconclusive. In vitro studies have shown that IFN-gamma has an effect on lymphocyte responses in addition to immunomodulatory effects on the monocyte/macrophage system. To investigate the in vivo effect of rHuIFN-gamma perioperatively on lymphocyte behavior in surgical patients, we studied 46 anergic patients undergoing major surgery. Treated patients (T, n = 24) received 100 microg rHuIFN-gamma subcutaneously (s.c.), and control patients (C, n = 22) received a placebo on preoperative days -7, -5, and -3 in a controlled, double-blinded placebo trial. Whole blood cultures were stimulated with mitogen on perioperative days, and cytokines were investigated in the supernatants. Interleukin-2 receptor (IL-2R) levels were significantly elevated in the treatment arm during the postoperative period (p < 0.05). The postoperative enhancement of IL-4 in C was completely attenuated in T (p < 0.05). IL-2 levels were elevated perioperatively in T but not in C. No significant effect of rHuIFN-gamma could be demonstrated on IL-10 or lymphocyte proliferation in vitro. From this pilot study, we conclude that preoperative in vivo immunomodulation of lymphocyte function with rHuIFN-gamma in anergic patients is effective. It improves immunoreactivity, as shown by elevated IL-2R levels. Elevated IL-2 and suppressed IL-4 levels indicate a shift toward a Th1-driven lymphocyte response.

Perioperative treatment with human recombinant interferon-gamma: a randomized double-blind clinical trial.

In spite of proven immunoregulatory effects in vitro of recombinant human interferon-gamma (rhIFN-gamma) in trauma, clinical trials remain inconclusive in such patients. To investigate the in vivo effect of rhIFN-gamma perioperatively in surgical patients we did a pilot study in 46 patients termed anergic by negative delayed-type hypersensitivity (DTH) skin test, who were undergoing major surgery (22 women and 24 men). They received 100 micrograms of rhIFN-gamma subcutaneously (treated [T]; n = 24) in a double-blind, placebo- (control [C]; n = 22) controlled manner on preoperative days -7, -5, and -3. Whole-blood cultures were stimulated on days -7, -1, 4, 7, and 10 for 12 h with or without LPS (1 microgram/mL). Mild side effects such as fever, headache, or chills were observed in 7/24 patients. No major complications occurred and no significant effect of rhIFN-gamma on HLA-DR, IL-1, and IL-8 was demonstrated. PGE2, TNF-alpha and IL-6 levels were elevated perioperatively in T. versus C. Neopterin, a metabolite of activated monocytes and macrophages, was significantly activated on days -1 (C: 7.6 +/- 1.2 versus T: 20.5 +/- 2.4 nmol/mL; P < 0.001), day 1 (C: 8.3 +/- 1.4 nmol/mL versus T: 24.9 +/- 2.8 nmol/mL; P < 0.001), and day 4 (C: 9.5 +/- 1.1 nmol/mL versus T: 16.0 +/- 1.8 nmol/mL; P < 0.05). Due to the overall lack of infectious complications during the investigation, no clinical effect was shown for rhIFN-gamma treatment. DTH skin testing failed to detect high-risk individuals in the patient population studied. In conclusion, we demonstrated in our pilot study that pre-operative immunomodulation with rhIFN-gamma in surgical anergic patients did not show severe side effects and modulated in vitro immunoresponse. A larger clinical trial in better-defined high-risk patients may show whether a reduction of infectious complications can be achieved.

Removal of mediators by continuous hemofiltration in septic patients.

Continuous hemofiltration currently represents standard renal replacement therapy in critically ill patients. Because higher ultrafiltration rates are related to better survival rates in experimental and clinical studies and hemofiltration results in fewer cardiovascular side effects than does conventional hemodialysis, the use of inflammatory mediator removal by this extracorporeal procedure has emerged. This article reviews clinically relevant principles of compound transport and the experimental and clinical effects of hemofiltration during sepsis. Hemofiltration did not have a major impact on plasma concentrations of prominent inflammatory cytokines (tumor necrosis factor-a and interleukins 1b, 6, and 8) and seems therefore not to be able to counterbalance endogenous cytokine production despite considerable cytokine removal in the filtrate. Contradictory results in the literature are discussed under the viewpoint of membrane-related sieving coefficients and plasma cytokine measurement. A significant reduction in plasma anaphylatoxin concentrations by hemofiltration is associated with impressive immunomodulatory and cardiodepressive ultrafiltrate effects. Thus far, however, the use of hemofiltration for nonrenal indications remains experimental and is not supported by controlled clinical trials. Modern strategies of blood purification that may be associated with a high degree of effectiveness for mediator removal (high-volume hemofiltration and heparin-induced extracorporeal lipoprotein-fibrinogen precipitation) are discussed.

Interferon-gamma modifies cytokine release in vitro by monocytes from surgical patients.

BACKGROUND: Treatment with interferon-gamma (IFN-gamma), a key mediator for adequate forward-regulatory monocyte immune capability, has been advocated to overcome posttraumatic mononuclear leukocyte paralysis. Conversely, IFN-gamma also is a potent proinflammatory mediator contributing to capillary leakage in sepsis-driven organ failure. The objective of this investigation was to further define the potential of IFN-gamma as a modifier of monocyte activity before and after injury. METHODS: Whole blood samples from 19 patients (7 female and 12 male patients; age, 68 +/- 5 years) before and after cardiac surgery with extracorporeal circulation were incubated under continuous rotation with lipopolysaccharide for 12 hours in the presence or absence of human recombinant IFN-gamma. Pro- and anti-inflammatory cytokines were determined in the plasma. RESULTS: Lipopolysaccharide-induced release of tumor necrosis factor-alpha, interleukin (IL)-6, IL-12, and IL-1Ra, and prostaglandin E2 was clearly augmented with IFN-gamma most strikingly postoperatively (p < 0.05). There was no effect on IL-1beta, neopterin, and soluble tumor necrosis factor-R release. CONCLUSION: Thus there is a wide spectrum of IFN-gamma activity on monocyte activation including anti-inflammatory properties. Since cellular preactivation facilitates monocyte reactivity toward IFN-gamma, we conclude that exogenous administration should be effective but must be carried out with great caution in patients with profound inflammation.

[Supportive sepsis therapy: what is still valid today?]. / Additive Sepsistherapie: Was bleibt heute?

Following several disappointing approaches to decrease the mortality of septic patients using various adjuncts, protein C is the first substance leading to a significant reduction of the mortality rate (PROWESS). The increased risk for bleeding complications in patients receiving protein C, however, limits its use in septic surgical patients. Administration of AT III in septic patients (KYBERSEPT) reduced the mortality rate solely in patients which were not treated with heparin. Although hydrocortisone reduced the amount of vasoactive katecholamines the effect of hydrocortisone administration on the survival rate remains unknown. In summary, early intervention to eliminate the septic focus and the use of antibiotics according to an antibiogramm still remains the most efficient basis for the treatment of septic patients.

Coagulation inhibitor replacement during sepsis: useless?

OBJECTIVE: Because coagulatory activation in sepsis is triggered mainly by tissue factor release from endothelial cells and blood monocytes during their activation via proinflammatory cytokines, inhibition of coagulation by exogenous administration of coagulation inhibitors has been proposed. These strategies should allow us to prevent and treat excessive coagulatory activation, thereby potentially preventing sepsis-induced organ dysfunction. Potential therapies include the natural coagulation inhibitors antithrombin, activated protein C, and tissue factor pathway inhibitor, as well as direct thrombin inhibition by recombinant hirudin. DATA SOURCES: A limited review of the published literature using all sources was undertaken. STUDY SELECTION: Selected clinical and experimental studies with coagulatory inhibitors were analyzed. CONCLUSIONS: The biological properties of coagulatory activation during sepsis (coagulation as a protective mechanism to control the septic focus, e.g., fibrin deposition during peritonitis) are not completely understood. Therefore, one has to be careful when administering coagulatory inhibitors, especially because patients with multiple organ dysfunction syndrome often do not show the widespread fibrin deposition in nutritive blood vessels that have been seen experimentally. How might these patients benefit from thrombin inhibition? Coagulatory activation per se seems unlikely to directly cause deterioration of organ function, although it is involved in generalized endothelial activation with consecutive mediator release and increased leukocyte-endothelial cell interaction. Antagonism of inflammatory mediators and, consecutively, endothelial cell activation might be a better target in adjunctive sepsis therapy, with improvement in septic microcirculatory disturbances. Administration of natural pleiotropic coagulation inhibitors that are documented positive effects on the microcirculation, (such as activated protein C, antithrombin) seems to be promising.

Hemofiltrate from patients with severe sepsis and depressed left ventricular contractility contains cardiotoxic compounds.

Myocardial dysfunction due to sepsis is common in patients with multiple organ dysfunction syndrome and is believed to be produced by inflammatory mediators. Some of these mediators may be eliminated by continuous hemofiltration, which is a standard procedure in an ICU for renal replacement therapy. This study was designed to directly compare the effects of ultrafiltrates from patients with sepsis (UFs) with ultrafiltrates from healthy volunteers (UFh) in well-characterized cardiomyocyte culture systems. Isovolemic hemofiltration (filtration rate: 2 L/h, polyamide membrane) was performed during 12 hours in 5 patients with severe sepsis (Elebute Score >20) and simultaneously reduced left ventricular contractility (left ventricular stroke work index [LVSWI] <30 g m/m2) and in 5 healthy volunteers. Inflammatory mediator concentrations (interleukin [IL]-1beta, IL-6, IL-8, tumor necrosis factor [TNF] alpha, C3a, and C5a) were measured in plasma and ultrafiltrate samples taken shortly after the beginning of the hemofiltration procedure. Cell culture experiments were done comparing UFs with UFh by using spontaneously beating or electrically driven neonatal rat cardiomyocyte cultures. UFs contained significantly higher amounts of IL-1, IL-8, and C3a when compared to UFh. Simultaneously, UFs induced a decrease in the contraction frequency of electrically-stimulated cardiomyocytes, whereas UFh had no effect. The cardiotoxic effect could be reversed by the addition of a high concentration (2.4 mM) of Ca++. Hemofiltration did not alter parameters of cardiac performance during 12 hours in patients with sepsis. UFs induced significant cardiotoxic effects in rat cardiomyocytes, whereas UFh showed no cardiotoxicity. Contact of blood with the hemofiltration membrane did not induce activation of cardiotoxic mediators. Significantly higher filtration rates may be required to improve left ventricular contractility in patients with sepsis by hemofiltration.

HMG-1 as a late mediator of endotoxin lethality in mice.

Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia). Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Here a potential late mediator of lethality is identified and characterized in a mouse model. High mobility group-1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1. Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure. Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal. Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target.

Evaluation of Fc-receptor positive (FcR+) and negative (FcR-) monocyte subsets in sepsis.

The monocyte/macrophage (Mphi is central in the regulation of the immune response in states of trauma and sepsis. Because monocyte subsets, characterized by expression of the Fc-receptor (FcR), were shown to play distinct immunologic roles in trauma, it was the objective of this study to assess insights into the functional role of FcR positive (FcR+) and negative (FcR-) subclasses in surgical sepsis. In a prospective study, peripheral blood Mphi from 20 septic patients and 10 healthy volunteers were evaluated on consecutive days after the onset of sepsis. FcR+/- subsets were separated by rosetting with antibody-coated human erythrocytes. Receptor expression and synthesis of proinflammatory cytokines were used to evaluate the functional role of these cells. We demonstrated a significant monocytosis (350%; p<.01) and suppression of human lymphocyte antigen (HLA-DR) expression (35%; p<.05). Synthesis of Interleukin-1beta (IL-1beta; e.g., Day 1: 230+/-30 pg/mL) and Interleukin-6 (IL-6; e.g., Day 1: 1920+/-350 U/mL) were significantly higher (p<.05) in FcR+ subsets than in controls (IL-1beta: 100+/-5 pg/mL; IL-6: 353+/-75 U/mL). Tumor necrosis factor-alpha (TNF-alpha) was elevated in FcR+ monocytes but did not reach a significant value. Interleukin-8 (IL-8) synthesis showed only on Day 1 and in controls significant differences in FcR+ and FcR- cells (Day1: FcR-: 19.6+/-4.1 nM; FcR+: 9+/-4.3 nM). Sepsis results in a significant shift toward FcR+ monocytes. This cell population is characterized by high proinflammatory cytokine synthesis. The extent of this shift seems to identify a group of high risk septic patients that might benefit from immunomodulatory therapy.

T-cell reactivity and its predictive role in immunosuppression after burns.

OBJECTIVE: To obtain further insight into the constitutional, phenotype-dependent changes of T-helper-1 and T-helper-2 signature lymphokine synthesis after trauma. DESIGN: Prospective, descriptive study. SETTING: Intensive care unit of a burn center in a community hospital. PATIENTS: Ten patients 1, 3, 5, and 7 days after major burn injury and 15 healthy individuals. INTERVENTIONS: Peripheral blood mononuclear cells were separated and incubated (5 hrs) for cytokine production induced by the accessory cell-independent stimulus of ionomycin and phorbol 12-myristate 13-acetate. After fixation and permeabilization, cell samples were immunofluorescently stained for cell surface antigens (CD4 and CD8), intracellular interferon (IFN)-gamma, and interleukin (IL)-4 synthesis. Results were correlated with corresponding enzyme-linked immunosorbent assay measurements of the culture supernatants. MEASUREMENTS AND MAIN RESULTS: The phenotypic analysis of the composition of the helper (CD4) and suppressor/cytotoxic (CD8) T-cell subset demonstrated that patients suffering from major burns and healthy controls express these antigens in similar percentages. The ratio of CD4 positive to CD8 positive/CD16 negative T-cell subsets showed no significant changes after trauma compared with controls. The production of IL-4 was excessively up-regulated while the release of IFN-gamma was only slightly increased. The predominant cell source of IL-4 after burn trauma was the CD8+ cell with nearly five-fold increased production on day 5 (7.2+/-2.6%) vs. 1.5+/-0.4% in controls. While CD8+ cells are also capable of enhancing their IFN-gamma synthesis under stress by about 60% due to the significant participation of the naive CD45RA+ subset, the CD4+ IFN-gamma release remained largely unchanged. With this study, we demonstrated that in nonsurvivors the number of CD8+ IL-4-producing cells was significantly higher compared with controls; also, the number of IFN-gamma-releasing memory/effector CD45RO+ cells was lower compared with survivors. CONCLUSIONS: In previous experiments, we show that a shift to T(H)2 dominated phenotypes increases the risk for postburn infection. The current study confirms that major burns induce a significant shift of cytokine response in the T(H)2 direction and demonstrates that the CD8+, rather than the CD4+ phenotype, is present. Increased IL-4 production is associated with the T(H)2 lymphocyte. These diagnostic tests may help to differentiate patients with compensatory anti-inflammatory response syndrome and immunosuppression from those patients in the proinflammatory state associated with the systemic inflammatory response syndrome. The profile described in this article is associated with immunosuppression and may contraindicate attempts at anti-inflammatory therapy for sepsis.

Immune dysfunction in trauma.

This article documents that all immunomodulation strategies for patients sustaining traumatic injury are still under intense investigation. Although we can speculate that combination strategies may be more beneficial than single-agent immunomodulation approaches, comprehensive clinical studies are required to determine efficacious immune therapy for trauma patients. The only strategy available to clinicians caring for trauma patients is immunonutrition, and this should be strongly considered as a rational approach to improve immune function and reduce septic complications in critically ill or injured patients.

Systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF): are we winning the battle?

The problems of inflammation and infection leading to organ dysfunction and failure continue to be the major problems after injury and operations and with intensive care for many diseases and problems. When SIRS goes to MODS and MOF, the mortality becomes high, ranging from 30-80% depending on the number of failed organs. In spite of this, there have been recent exciting discoveries and contributions to patient care. A reasonable question then is, are we making progress and if so, can we document it? Are the incidence and mortality of MOF decreasing? The literature comparing care over some years suggests a decrease in ICU mortality in patients with severe organ failure, a decrease in elective surgical mortality, and improvement in the results of care and outcome for trauma patients. Review of problems occurring in sick and injured patients indicates that certain problems are decreasing in frequency, such as renal failure and ARDS after trauma, stress gastrointestinal bleeding, and abdominal abscesses, and these should improve outcome. There are a number of exciting therapies that help certain patients but not everyone. These controversies challenge us to focus on where and when there are positive benefits. Risk factors for MOF are addressed to focus on early intervention. The possibilities of multiple therapeutic agents are described. Finally, we describe and emphasize our recommendation to strive to prevent MODS and SIRS.

Therapeutic immunomodulatory approaches for the control of systemic inflammatory response syndrome and the prevention of sepsis.

In the sequelae of massive traumatic stress, substantial impairment of immunologic reactivity has been demonstrated to correlate clinically with increased susceptibility to serious infection. Posttraumatic immune abnormalities consist basically of two coexistent mechanisms: Hyperinflammation and depression of cell-mediated immune responses. It is our understanding that the endogenous ability of the organism to survive overwhelming trauma is insufficient and requires exogenous support to prevent the conversion from systemic inflammatory response syndrome to bacterial sepsis and septic shock. The objectives of immunomodulatory interventions, which should be started as early as possible after tissue destruction, include a) prevention of excessive macrophage stimulation via neutralization of circulating endotoxins and exotoxins with high doses of polyvalent immunoglobulin and soluble complement receptors, b) global short-term (<72 hrs) down-regulation of inflammatory monocyte/macrophage and polymorphonuclear neutrophil activity, and c) restoration of cell-mediated immune performance to overcome posttraumatic functional paralysis. Among recent promising strategies, the use of granulocyte-macrophage colony-stimulating factor, pentoxifylline, and recombinant human interleukin-13 has been suggested, all of them predominantly down-regulating the Mphi (monocyte/macrophage) inflammatory potential. Cyclooxygenase inhibitors such as indomethacin and thymomimetic peptides can help normalize the immunoreactivity by restoring the forward-regulatory pathway of cell-mediated immunity responses. The efficacy of interferon to reduce infection and deaths in severely injured patients has been assessed in clinical trials. Still other compounds, i.e., CNI-1493, interleukin-11, tissue factor pathway inhibitors, and PGG-Glucan represent auspicious immunomodulatory approaches for control of posttraumatic or postoperative infections.