Binding characteristics of [3H]-JSM10292: a new cell membrane-permeant non-peptide bradykinin B2 receptor antagonist.

BACKGROUND AND PURPOSE: A (3) H-labelled derivative of the novel small-molecule bradykinin (BK) B(2) receptor antagonist JSM10292 was used to directly study its binding properties to human and animal B(2) receptors in intact cells and to closely define its binding site. EXPERIMENTAL APPROACH: Equilibrium binding, dissociation and competition studies with various B(2) receptor ligands and [(3) H]-JSM10292 were performed at 4°C and 37°C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B(2) receptors of human and animal origin. KEY RESULTS: [(3) H]-JSM10292 bound to B(2) receptors at 4°C and at 37°C with the same high affinity. Its dissociation strongly depended on the temperature and increased when unlabelled B(2) receptor agonists or antagonists were added. [(3) H]-JSM10292 is cell membrane-permeant and thus also bound to intracellular, active B(2) receptors, as indicated by the different 'nonspecific' binding in the presence of unlabelled JSM10292 or of membrane-impermeant BK. Equilibrium binding curves with [(3) H]-JSM10292 and competition experiments with unlabelled JSM10292 and [(3) H]-BK showed a different affinity profile for the wild-type B(2) receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B(2) receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B(2) receptor ligand. CONCLUSIONS AND IMPLICATIONS: [(3) H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B(2) receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B(2) receptors.

TNF-α respecifies human mesenchymal stem cells to a neural fate and promotes migration toward experimental glioma.

Bone marrow-derived human mesenchymal stem cells (hMSCs) have become valuable candidates for cell-based therapeutical applications including neuroregenerative and anti-tumor strategies. Yet, the molecular mechanisms that control hMSC trans-differentiation to neural cells and hMSC tropism toward glioma remain unclear. Here, we demonstrate that hMSCs incubated with 50 ng/ml tumor necrosis factor alpha (TNF-α) acquired astroglial cell morphology without affecting proliferation, which was increased at 5 ng/ml. TNF-α (50 ng/ml) upregulated expression of numerous genes important for neural cell growth and function including LIF (leukemia inhibitory factor), BMP2 (bone morphogenetic protein 2), SOX2 (SRY box 2), and GFAP (glial fibrillary acidic protein), whereas NES (human nestin) transcription ceased suggesting a premature neural phenotype in TNF-α-differentiated hMSCs. Studies on intracellular mitogen-activated protein kinase (MAPK) signaling revealed that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activity abolished the TNF-α-mediated regulation of neural genes in hMSCs. In addition, TNF-α significantly enhanced expression of the chemokine receptor CXCR4 (CXC motive chemokine receptor 4), which facilitated the chemotactic invasiveness of hMSCs toward stromal cell-derived factor 1 (SDF-1) alpha. TNF-α-pretreated hMSCs not only exhibited an increased ability to infiltrate glioma cell spheroids dependent on matrix metalloproteinase activity in vitro, but they also showed a potentiated tropism toward intracranial malignant gliomas in an in vivo mouse model. Taken together, our results provide evidence that culture-expansion of hMSCs in the presence of TNF-α triggers neural gene expression and functional capacities, which could improve the use of hMSCs in the treatment of neurological disorders including malignant gliomas.

Observable effects of mechanical stress induced by sample spinning in solid state nuclear magnetic resonance.

The stress-induced change in chemical shielding induced by sample spinning is measured and interpreted theoretically. By considering the rotating sample as an elastic body in the plane-strain approximation, the internal stress field as a function of sample size, rotation frequency, and elastic constants is determined. This stress field and the dependence of chemical shielding on strain, as determined by first-principles calculations, are combined to predict the shielding dependence on rotation frequency under isothermal conditions in single crystal gallium phosphide. The prediction is in good qualitative agreement with the experiment. Little to no effect is detected in powder samples of both gallium phosphide and copper iodide, and it is argued that this follows from the stress distribution in granular material, as opposed to bulk crystals. Finally, the temperature and pressure dependence of the chemical shielding is estimated from these considerations and found consistently to underestimate the experimental values, indicating the importance of finite-temperature anharmonic effects even in very simple solids.

Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells.

Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.

Hemofiltration does not influence early S-100B serum levels in septic shock patients receiving stress doses of hydrocortisone or placebo.

BACKGROUND: The prognosis in patients with hyperdynamic septic shock correlates with the presence and the severity of septic encephalopathy. However, the neurological evaluation is considerably influenced by the use of analgesia sedation during mechanical ventilation. An early concentration peak of the neuroprotein S-100B in serum reflects both cellular damage at an increased permeability of the blood-brain-barrier and a delayed renal elimination. Thus, the objective of this study was to analyze the effect of continuous veno-venous hemofiltration (CVVH) on early S-100B serum levels in septic shock patients, who were treated with either stress doses of hydrocortisone or placebo. METHODS: Twenty-four consecutive patients, who met the ACCP / SCCM criteria for septic shock, were enrolled in this prospective, randomised, double-blind, single-center trial. The severity of illness at recruitment was graded using the APACHE II and SAPS II scoring systems; the MODS was described by the SOFA score. All patients were prospectively randomised to receive either stress doses of hydrocortisone or placebo. Hydrocortisone was started in 12 patients with a loading dose of 100 mg and followed by a continuous infusion of 0.18 mg/kg/h for 6 days. RESULTS: Median S-100B serum levels of the hydrocortisone group decreased from 0.32 ng/ml (0.19-3.60) at study entry to 0.07 ng/ml (0.04 - 0.32) 6 days later without significant differences compared to the placebo group. Patients undergoing CVVH showed significantly higher S-100B serum values compared to patients without CVVH (p<0.001). However, initial median S-100B serum levels of the CVVH group even increased from 0.92 ng/ml (0.16 - 4.63) to 2.33 ng/ml (0.59 - 2.44) 30 hours after study entry, reaching data ranges already known in patients with out-of-hospital cardiac arrest or severe traumatic brain injury. CONCLUSION: Early S-100B serum levels in septic shock patients receiving either stress doses of hydrocortisone or placebo were not influenced by CVVH. For the first time, we observed a similar extent of S-100B serum increase in CVVH patients, who had significantly higher S-100B serum values compared to those without CVVH, as reported for out-of-hospital cardiac arrest or severe traumatic brain injury. Hypercortisolemia induced by the infusion of stress doses of hydrocortisone did not significantly reduce early S-100B serum concentrations with time.

Hemofiltrastion does not influence early S-100B serum levels in septic shock patients receiving stress doses of hydrocortisone or placebo.

BACKGROUND: The prognosis in patients with hyperdynamic septic shock correlates with the presence and the severity of septic encephalopathy. However, the neurological evaluation is considerably influenced by the use of analgesia sedation during mechanical ventilation. An early concentration peak of the neuroprotein S-100B in serum reflects both cellular damage at an increased permeability of the blood-brain-barrier and a delayed renal elimination. Thus, the objective of this study was to analyze the effect of continuous veno-venous hemofiltration (CVVH) on early S-100B serum levels in septic shock patients, who were treated with either stress doses of hydrocortisone or placebo. METHODS: Twenty-four consecutive patients, who met the ACCP / SCCM criteria for septic shock, were enrolled in this prospective, randomised, double-blind, single-center trial. The severity of illness at recruitment was graded using the APACHE II and SAPS II scoring systems; the MODS was described by the SOFA score. All patients were prospectively randomised to receive either stress doses of hydrocortisone or placebo. Hydrocortisone was started in 12 patients with a loading dose of 100 mg and followed by a continuous infusion of 0.18 mg/kg/h for 6 days. RESULTS: Median S-100B serum levels of the hydrocortisone group decreased from 0.32 ng/ml (0.19-.60) at study entry to 0.07 ng/ml (0.04-0.32) 6 days later without significant differences compared to the placebo group. Patients undergoing CVVH showed significantly higher S-100B serum values compared to patients without CVVH (p>0.001). However, initial median S-100B serum levels of the CVVH group even increased from 0.92 ng/ml (0.16 - 4.63) to 2.33 ng/ml (0.59-2.44) 30 hours after study entry, reaching data ranges already known in patients with out-of-hospital cardiac arrest or severe traumatic brain injury. CONCLUSION: Early S-100B serum levels in septic shock patients receiving either stress doses of hydrocortisone or placebo were not influenced by CVVH. For the first time, we observed a similar extent of S-100B serum increase in CVVH patients, who had significantly higher S-100B serum values compared to those without CVVH, as reported for out-of-hospital cardiac arrest or severe traumatic brain injury. Hypercortisolemia induced by the infusion of stress doses of hydrocortisone did not significantly reduce early S-100B serum concentrations with time.

[3D-Culturing of human osteoblastic cells with vessel like nutrient supply]. / 3D-Kultivierung humaner osteoblastärer Zellen unter gefässähnlicher Nährstoffversorgung.

AIM: The treatment of large, critical-size bone defects is a major therapeutic problem in orthopaedic and reconstructive surgery. The engineering of bone tissue could be used to replace lost bone mass. However, scaffolds seeded with vital cells and cultured in vitro suffer from poor oxygen and nutrient supply centrally, when the constructs exceed a critical volume. Therefore, we have established an osteoblastic cell culture in a new 3D-culture chamber with an artificial, vessel-like central membrane, allowing continuous nutrient supply. METHOD: Human osteoblasts were cultured in a 3D-like manner using a perfusion chamber for one week. In this system, the nutrient supply is guaranteed by a vessel-like, semipermeable polysulfone membrane with a continuous flow of medium. After fixation and cryosectioning, histological and immunohistological staining and scanning electron microscopy was carried out. RESULTS: Examinations reveal 3D cell growth around the central vessel. Formation of an extracellular matrix, rich in collagen type I and fibronectin, was detected immunohistochemically. Furthermore, we demonstrated cell adherence to the membrane and examined the surface morphology by scanning electron microscopy. CONCLUSION: The innovative approach for 3D-culturing of human osteoblasts in a system with a central nutrient supply opens up new possibilities for the in vitro cultivation for tissue engineering.

[Identification of high-risk patients after minor craniocerebral trauma. Measurement of nerve tissue protein S 100]. / Identifikation von Hochrisikopatienten nach leichtem Schädel-Hirn-Trauma. Messung des neuroglialen Proteins S100.

The indication for an initial cranial computed tomography (CCT) in minor head trauma (MHT) patients remains the subject of discussion. The aim of this study was to investigate whether a newly developed, rapid test system (ELECSYS S100, Roche Diagnostics) might allow a diagnostically valid, reproducible measurement of S 100 in MHT patients. Blood samples were drawn from 75 MHT patients, a CCT scan was performed, and those with a post-traumatic intracranial lesion counted as CCT+. Results were compared to a healthy control group (n=17). Of the 75 patients included in the study, 14 were stratified as CCT+. The systemic concentration of S 100 in these CCT+ patients was significantly increased (0.31 microg/l) compared to the healthy control group (0.04 microg/l) as well as to the CCT-negative patients (0.08 microg/l). The ELECSYS S100 system allows a rapid, valid, and reproducible assessment of S 100B in patient serum and this concentration is significantly elevated in patients suffering from intracranial lesions as shown by initial CCT scan. Hence, this study is the basis for a multicenter trial currently underway to confirm the results of our pilot study.

Initial RNA expression in human monocytes after multiple injury: a screening pilot study on potentially trauma-sensitive factors by using the microarray-technique.

BACKGROUND: Pathological affection of the immune system is one of the initiating mechanisms for the induction of multiple organ failure (MOF) in patients suffering from multiple injuries. Potential responsible intracellular mechanisms such as initial monocyte mRNA expression of specific mediators remain poorly studied, so far. Hence, we applied the microarray technique for screening of a wide variety of genes in circulating monocytes of multiple injured patients and compare the molecular results to the clinical course of the patients (MOF-score). METHODS: In our prospective pilot study 6 patients were enclosed presenting with blunt multiple injuries (Injury Severity Score 16 to 57 points). Monocytes were isolated out of sequentially drawn samples (6, 12, 24 and 48 hours after trauma) using magnetic cell sorting (CD14) and a human microarray system was used (Atlas stress 1.2, Clontech, 1176 genes). Alterations in the sequential samples were identified by calculating ratios to baseline levels on admission and cluster analysis was performed (Spotfire Decision). RESULTS: Only 86 (ca.5%) genes displayed an obvious signal. The house-keeping genes clustered well together in all patients in contrast to a substantial inter-individual variability of the other signal giving genes. No mediator burst of the classical pro- or anti-inflammatory cascade were detected. CONCLUSION: We demonstrate for the first time a screening analysis of mRNA expression patterns in circulating monocytes of multiple injured patients indicating that only very few genes appeared to be influenced by the traumatic event. So far, no correlation to the severity of trauma or MOF could be detected.

Serum S-100B protein levels in young amateur soccer players after controlled heading and normal exercise.

BACKGROUND: Repeated impacts by heading might lead to significant head injuries in soccer players comparable to those of patients with accidental minor traumatic brain injury (TBI). The neuroprotein S-100B released into the circulation is suggested to be a reliable marker indicating brain damage. The objective was to evaluate the neuroprotein S-100B serum levels in young amateur soccer players early after controlled heading compared to early measurements after normal exercise as well as in other patients after minor TBI. MATERIAL AND METHODS: Sixty-one male amateur soccer players (median age 15.3 years) performed controlled heading aimed at the forehead for 55 minutes. Data were compared to 58 male amateur soccer players (15.9 years) performing 61 minutes of normal exercise training without head contact and 81 young male patients early after TBI who underwent computed tomography (CCT) for detection of intracranial lesions. First venous blood samples were drawn before the training sessions, second and third samples 60 and 360 minutes after heading or 64 and 355 minutes after exercise, respectively, 65 and 366 minutes after TBI. RESULTS: Median S-100B serum levels of the heading group only slightly increased from 0.15 ng/ml to 0.18 ng/ml 60 minutes after end of training. Within 360 minutes S-100B values decreased again to 0.15 ng/ml reaching the initial values. S-100B serum values of the exercise group showed a similar transient increase exhibiting significant lower levels before exercise (0.10 ng/ml) as well as 64 minutes (0.11 ng/ml) and 355 minutes after exercise (0.09 ng/ml) compared to the heading group. According to age stratification in the heading group, starting median S-100B levels were significantly higher in subjects with 12-13 years (0.22 ng/ml) and 14-15 years (0.17 ng/ml) compared to those with 16-17 years of age (0.06 ng/ml). None of the subcollectives did reach median S-100B levels of the CCT+ group (n = 20) at admission (0.62 ng/ml) or 366 minutes later (0.32 ng/ml), which were significantly elevated compared to those of the CCT group (n = 61) at admission (0.10 ng/ml) or 370 minutes later (0.08 ng/ml). CONCLUSIONS: Controlled repetitive heading in young amateur soccer players leads to a transient increase between 60 to 360 min after training, but does not appear to evoke a longer lasting S-100B release into serum indicating cellular brain damage. After heading S-100B levels are significantly elevated compared to normal exercise. Although soccer players with 12-13 years and 14-15 years revealed significantly higher S-100B values than with 16-17 years of age, the transient increase is independent of the age-related starting values. However, since brain damage due to abrupt heading a ball of high speed or accidental trauma during regular soccer games cannot be excluded, S-100B measurements in soccer players are initiated during video-controlled soccer games.

Elevated expression of polymorphonuclear leukocyte elastase in breast cancer tissue is associated with tamoxifen failure in patients with advanced disease.

Besides a variety of other proteases, polymorphonuclear leukocyte elastase (PMN-E) is also suggested to play a role in the processes of tumour cell invasion and metastasis. Yet, there is only limited data available on the relation between the tumour level of PMN-E and prognosis in patients with primary breast cancer, and no published information exists on its relation with the efficacy of response to systemic therapy in patients with advanced breast cancer. In the present study, we have measured with enzyme-linked immunosorbent assay the levels of total PMN-E in cytosolic extracts of 463 primary breast tumours, and have correlated their levels with the rate and duration of response on first-line tamoxifen therapy (387 patients) or chemotherapy (76 patients) in patients with locally advanced and/or distant metastatic breast cancer. Furthermore, the probabilities of progression-free survival and postrelapse survival were studied in relation to the tumour levels of PMN-E. Our results show that in logistic regression analysis for response to tamoxifen treatment in patients with advanced disease, high PMN-E tumour levels were associated with a poor rate of response compared with those with low PMN-E levels (odds ratio: OR, 0.40; 95% CI, 0.22-0.73; P=0.003). After correction for the contribution of the traditional predictive factors in multivariate analysis, the tumour PMN-E status was an independent predictor of response (P=0.01). Furthermore, a high tumour PMN-E level was related with a poor progression-free survival (P<0.001) and postrelapse survival (P=0.002) in a time-dependent analysis. In contrast, the tumour level of PMN-E was not significantly related with the efficacy of response to first-line chemotherapy in patients with advanced breast cancer. Our present results suggest that PMN-E is an independent predictive marker for the efficacy of tamoxifen treatment in patients with advanced breast cancer.

Systemic regulation of distraction osteogenesis: a cascade of biochemical factors.

This study investigates the systemic biochemical regulation of fracture healing in distraction osteogenesis compared with rigid osteotomy in a prospective in vivo study in humans. To further clarify the influence of mechanical strain on the regulation of bone formation, bone growth factors (insulin-like growth factor [IGF] I, IGF binding protein [IGFBP] 3, transforming growth factor [TGF] beta1, and basic FGF [bFGF]), bone matrix degrading enzymes (matrix-metalloproteinases [MMPs] 1, 2, and 3), human growth hormone (hGH), and bone formation markers (ALP, bone-specific ALP [BAP], and osteocalcin [OC]) have been analyzed in serum samples from 10 patients in each group pre- and postoperatively. In the distraction group, a significant postoperative increase in MMP-1, bFGF, ALP, and BAP could be observed during the lengthening and the consolidation period when compared with the baseline levels. Osteotomy fracture healing without the traction stimulus failed to induce a corresponding increase in these factors. In addition, comparison of both groups revealed a significantly higher increase in TGF-beta1, IGF-I, IGFBP-3, and hGH in the lengthening group during the distraction period, indicating key regulatory functions in mechanotransduction. The time courses of changes in MMP-1, bone growth factors (TGF-beta1 and bFGF), and hGH, respectively, correlated significantly during the lengthening phase, indicating common regulatory pathways for these factors in distraction osteogenesis. Significant correlation between the osteoblastic marker BAP, TGF-beta1, and bFGF suggests strain-activated osteoblastic cells as a major source of systemically increased bone growth factors during callus distraction. The systemic increase in bFGF and MMP-1 might reflect an increased local stimulation of angiogenesis during distraction osteogenesis.

Rapid identification of high-risk patients after minor head trauma (MHT) by assessment of S-100B: ascertainment of a cut-off level.

BACKGROUND: Elevated blood S-100B levels were described by several authors for reliable identification of patients with intracerebral complications after minor head trauma (MHT). Yet, test systems used so far require more than 3 hours processing period which is too long to enable immediate further diagnostic or therapeutic consequences. Therefore we validate a new rapid test version for S-100B measurements and established an effective cut-off level to identify high risk patients. METHODS: 104 patients suffering from MHT were enrolled. After taking blood samples S-100B values were achieved by the long-term and rapid (40 min processing time) test system, respectively, and compared using linear regression analysis. For determination of an effective cut-off level receiver operating characteristics curves were calculated in accordance with cranial computed tomography findings. RESULTS: S-100B concentrations correlated significantly using both test systems. A cut-off level of 0.18ng/ml was calculated in plasma samples. CONCLUSIONS: S-100B concentrations above the cut-off level measured within 40 min after blood sampling allows safe identification and immediate treatment of intracerebral lesions (e.g. epidural and subdural hematoma, subarachnoid hemorrhage, diffuse brain edema etc.) in MHT patients.

Effect of C1-esterase-inhibitor on capillary leak and inflammatory response syndrome during arterial switch operations in neonates.

OBJECTIVE: To determine if prophylactic administration of C1-esterase-inhibitor would have a beneficial effect on postoperative weight gain and the inflammatory response in neonates undergoing cardiac surgery with cardiopulmonary bypass (CPB). DESIGN: Randomized, double-blinded study. SETTING: University-affiliated heart center. PARTICIPANTS: Twenty-four neonates with transposition of the great arteries. INTERVENTIONS: In group inhibitor (INH) patients (n = 12), 100 IU/kg of C1-esterase-inhibitor (Berinert) was given 30 minutes before CPB. In group placebo (P) patients (n = 12), placebo was administered instead. Interleukin (IL)-6, C3a anaphylatoxin, C1 activity, prekallikrein, Hageman factor, D-dimers, and clinical parameters were measured 6 times perioperatively. MEASUREMENTS AND MAIN RESULTS: All 24 patients had an uneventful clinical course. Mean arterial pressure and pulmonary oxygenation after CPB were superior in group INH patients. The weight gain on postoperative days 1 to 4 was significantly less in group INH patients compared with group P (55 +/- 59 g vs. 340 +/- 121 g, day 1). The concentration of IL-6 (76 +/- 17 pg/mL vs. 262 +/- 95 pg/mL during CPB) was significantly lower in group INH patients compared with group P patients. In contrast, no influence on C3a anaphylatoxin and coagulation factors was found. CONCLUSION: Prophylactic application of C1-esterase-inhibitor in neonates undergoing arterial switch operations produces less inflammatory response compared with placebo. This difference may have contributed to improved clinical parameters, including less weight gain postoperatively.

Elevated serum levels of S-100B reflect the extent of brain injury in alcohol intoxicated patients after mild head trauma.

Elevated systemic levels of S-100B are proposed as a potential indicator of brain damage in identifying high-risk patients after mild head trauma (MHT). Although incidence of alcohol intoxication is high in these patients, the influence of alcohol intoxication on S-100B levels is unclear. Therefore, the aim of our study was to investigate serum concentrations of S-100B in intoxicated (group 1) and sober (group 2) patients after MHT in comparison with those of mild (group 3) or severely intoxicated (group 4) individuals without trauma. S-100B was significantly increased in MHT patients exhibiting posttraumatic lesions in initial cranial computed tomography scan. Alcohol intoxication did not elevate S-100B levels in group 3 or 4 subjects. Our data indicate for the first time that alcohol intoxication does not influence the diagnostic value of S-100B measurements in patients after MHT.

Early cellular brain damage and systemic inflammatory response after cardiopulmonary resuscitation or isolated severe head trauma: a comparative pilot study on common pathomechanisms.

Severe neurological deficits are common characteristics of patients surviving cardiopulmonary resuscitation (CPR) or isolated severe head trauma (SHT). For comparative evaluation of underlying pathomechanisms, 22 patients with out-of-hospital cardiac arrest and successful CPR as well as 10 patients with SHT were included in our prospective study. Circulating S-100B was determined as an indicator of cellular brain damage. Interleukin-8 (IL-8), soluble E-selectin (sE-selectin) and polymorphonuclear (PMN-) elastase were measured as markers of systemic inflammation following whole body ischaemia and reperfusion injury. Venous blood samples were drawn on scene (median time 11.0 min after starting basic life support) and in the intensive care unit (median time 12.5 h thereafter) in CPR patients and at admission to hospital (median time 43.8 min after trauma) and approx. 12 h later in SHT patients. Biochemical parameters in these samples were compared with specimens taken from 20 healthy volunteers. Initial median S-100B levels of the CPR and SHT patients were both significantly increased compared with the controls. Twelve hours later, significant falls in S-100B revealed no differences between the two patient groups, but did not reach control values. Median IL-8 and sE-selectin levels entry to the study were elevated in both patient groups compared with controls and showed further rises within the following 12 h. Finally, increased initial median levels of PMN-elastase revealed significant differences between the patient groups and between patients and controls. Twelve hours later, median PMN-elastase values were equally elevated in the CPR and SHT subjects. Our preliminary data suggest similar pathomechanisms occurring after both CPR and SHT. Both clinical entities seem to be associated with early transient cellular brain damage as shown by prolonged rapidly increasing and subsequent fall in S-100B serum levels. In contrast, the prolonged elevation of circulating IL-8, sE-selectin and PMN-elastase may indicate a very similar systemic inflammatory response by endothelial cells and neutrophils initiated by ischaemia and reperfusion injury in both conditions. Further studies should be carried out to determine the cause and the prognostic value of these biochemical parameters in relation to long-term neurological outcome.

S-100b, sE-selectin, and sP-selectin for evaluation of hypoxic brain damage in patients after cardiopulmonary resuscitation: pilot study.

S-100b is thought to be a screening marker of hypoxic brain damage in patients with cardiac arrest. However, the time-dependent occurrence and relevance of increased S-100b serum levels in out-of-hospital patients with cardiopulmonary resuscitation (CPR) is still discussed. The purpose of our study was to evaluate the diagnostic utility of S-100b measurements in comparison to that of adhesion molecules sE-selectin and sP-selectin in patients with CPR. Sixteen out-of-hospital patients (median age 69.6 years; range 59.2-82.2 years) suffering from cardiac arrest due to ventricular fibrillation, asystole, or electromechanical dissociation were recruited prospectively. Blood samples were drawn on scene after the return of spontaneous circulation (ROSC) and 12 hours after successful CPR. The reference group consisted of 10 patients with isolated severe head trauma (SHT) (Glasgow Coma Score

The effect of trauma on neutrophil L-selectin expression and sL-selectin serum levels.

Among identified adhesion molecules, the L-selectin on neutrophils enables the first step of leukocyte adherence to activated endothelial cells. To allow firm adhesion of neutrophils, L-selectin is then split off the cell membrane. It was hypothetized that an increase of the constitutively high serum level of soluble L-selectin may indicate an ongoing pathological neutrophil sequestration to the endothelial cells associated with activation and injury of the cells. To evaluate this hypothesis, sL-selectin serum levels and neutrophil L-selectin expression of healthy volunteers (group A, n = 15), as well as of surgical patients, were investigated. Group B (n = 26) included patients subjected to elective limb surgery (mean operation time, 122 min), and group C (n = 45) comprised trauma patients. sL-selectin serum levels were measured daily over a 14-day period. Neutrophil L-selectin expression was evaluated by FACS analysis using the humanized anti-L-selectin antibody HuDreg 55 over a period of 3 days at minimum in both experimental groups. The binding of sL-selectin to endothelial cells was also examined in vitro. Elective limb surgery resulted in lower pre- and post-operative sL-selectin plasma levels (800-1,000 ng/mL) compared to healthy volunteers (1,100-1,200 ng/mL) with insignificant changes throughout the study period. Trauma patients revealed even lower sL-selectin levels (400-600 ng/mL). When these patients were discriminated by the multiple organ dysfunction (MOD) score of Moore in +MOD (n = 9, ISS = 31.7) and -MOD (n = 36, ISS = 25.0), a significant difference became evident. In +MOD patients sL-selectin levels remained on a low basis of 350 ng/mL, whereas in -MOD patients the initial low sL-selectin level subsequently rose to 800 ng/mL, similar to that of elective surgery patients. FACS analysis revealed a significant drop in neutrophil L-selectin expression 24 h after trauma compared to normal. Also, +MOD and -MOD patients were significantly discriminated by the L-selectin expression at this time. The in vitro studies revealed evidence for binding of sL-selectin to endothelial cells independently on the presence of neutrophils. According to our data, increasing severity of the post-operative/posttraumatic course is associated with decreasing sL-selectin serum levels and also reduced neutrophil L-selectin expression. In view of the in vitro results, this probably indicates competitive enhanced binding of sL-selectin to endothelial cells, thus masking the elevated activation of neutrophils and their ability for endothelial adherence.

Immunomodulation in septic shock: hydrocortisone differentially regulates cytokine responses.

Cortisol is known to be an immunomodulatory hormone that exerts suppressive and permissive effects on the immune response. Little is known regarding the evolution of the cytokine response in human septic shock in the presence of hypercortisolemia induced by infusion of stress doses of hydrocortisone. Twenty-four consecutive patients with high-out-put circulatory failure (cardiac index, >4 liters/min per m(2)) who met the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee criteria for septic shock were enrolled in a prospective, double-blind study. The severity of illness at the time of enrollment was graded using the Acute Physiology and Chronic Health Evaluation II system, and the evolution of sepsis-induced organ dysfunction syndrome was assessed using Sepsis-Related Organ Failure Assessment scores. After randomization, hyper-cortisolemia was induced in 12 patients by infusion of 100 mg of hydrocortisone, followed by continuous infusion of 0.18 mg/kg per h. Levels of the circulating cytokines tumor necrosis factor alpha (TNF), interleukin 6 (IL-6), IL-8, and IL-10 were serially measured at prospectively defined time points during the first 5 d after randomization. The infusion of hydrocortisone was associated with significant reductions in serum IL-6 and IL-8 levels and with earlier resolution of the sepsis-induced organ dysfunction syndrome. IL-6 levels started to differ between the groups on day 5. The TNF and IL-10 responses were not altered by hydrocortisone infusion. Hydrocortisone infusion in septic shock differentially regulated the cytokine responses. IL-6 and IL-8 levels decreased significantly and IL-6 levels differed between the groups, whereas TNF and IL-10 levels were not affected by hydrocortisone. Stress doses of hydrocortisone may be a valuable immunomodulatory therapy for septic shock.