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.

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.

[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.

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.

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.

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.

Determination of plasma aprotinin levels by functional and immunologic assays.

We compared a functional (amidolytic) and an enzyme-linked immunosorbent assay (ELISA) method for determining aprotinin concentration in 82 plasma samples obtained from patients undergoing cardiac surgery with aprotinin therapy. There was good correlation between methods (r = 0.87); however, aprotinin measurements by chromogenic assay were significantly higher than by ELISA [234 +/- 104 kallikrein inhibitory units (KIU)/ml versus 155 +/- 88 KIU/ml; P = 0.0001]. This appeared to be attributable to differences in the potency of the material used to standardize the assays. When results were corrected to allow for potency of the standard, there was no significant difference between chromogenic and ELISA methods (234 +/- 104 KIU/ml versus 240 +/- 137 KIU/ ml), although the ELISA results tended to be higher in some samples. These data suggest that aprotinin concentrations measured by these methods cannot be used interchangeably, and care must be taken when interpreting data from studies measuring aprotinin.

Is perioperative plasma aprotinin concentration more predictable and constant after a weight-related dose regimen?

UNLABELLED: To determine whether a weight-related dose had advantages over a fixed, large-dose regimen, we measured plasma concentrations of aprotinin by using an enzyme-linked immunosorbent assay method at set time points in 30 patients having heart surgery with cardiopulmonary bypass. A weight-related dose comprising a preincision bolus injection of 40,000 kallikrein-inhibiting units (KIU)/kg (5.6 mg/kg) with the same amount given in the oxygenator prime was compared with a large-dose regimen of 2 x 10(6) KIU (280 mg) preincision bolus and addition to prime, together with an infusion of 500,000 KIU/h (70 mg/h). Peak plasma concentration in the Weight-Related group was less variable than with the fixed-dose regimen. Forty percent of patients allocated to the fixed-dose regimen had an aprotinin concentration of more than 400 KIU/mL, compared with none in the Weight-Related group; this suggests a relative overdosing in the early surgical period in the Fixed-Dose group. There was great individual variability between patients in the time-concentration curves for aprotinin, with no difference between the two regimens. The weight-related dose regimen benefited by not requiring an intraoperative infusion while achieving the same plasma concentrations of aprotinin. IMPLICATIONS: Peak plasma concentrations of aprotinin were less variable with a weight-related dose schedule. This has implications for safety with regard to control of anticoagulation and cost in patients with small body mass. Plasma concentrations varied greatly with time between patients. This observation has implications for determining an optimal dose on the basis of aprotinin's currently known mechanisms of action.

Evaluation of S-100b as a specific marker for neuronal damage due to minor head trauma.

Management of patients with minor head trauma (MHT) continues to be debated in the literature. Measurement of S-100b in serum has been introduced into the discussion as an additional screening tool for intracerebral injuries because routine cranial computed tomography (CCT) of a large number of patients causes logistic difficulties, and the neurologic examination is often impaired by a high frequency of coincidental intoxication. The aim of our study was to determine the diagnostic value of measuring S-100b in the serum of MHT patients to identify risk groups. Additional validity should be aquired by a comparison with plasma levels of polymorphonuclear neutrophil (PMN) elastase an established general trauma marker. A series of 52 patients with MHT were included in the prospective study. At admission the patients underwent a routine CCT scan to detect intracerebral lesions, and blood samples were drawn to investigate circulating levels of S-100b and PMN elastase. For comparison, data for a positive control group of 10 severe head trauma patients (initial Glasgow Coma Scale score < 8) and for a negative control group with 20 healthy volunteers were obtained. The interval between MHT and admission to our hospital was 73.4 +/- 47.0 minutes. The initial S-100b serum levels of MHT patients were 0.470 +/- 0.099 ng/ml, those of the positive control group were 7.16 +/- 3.77 ng/ml, and those of the negative control group were 0.05 +/- 0.01 ng/ml. Relevant pathologic CCT scans were detected in 28.8% of MHT patients; one patient of that group was subjected to immediate surgical intervention (1.9%). At a cut-off point of 0.1 ng/ml, the sensitivity of positive S-100b levels reached 100% and the specificity 40.5%. Plasma levels of PMN elastase reached 60.52 +/- 10.75 ng/ml in the MHT group, 66.4 +/- 14.92 ng/ml in the severely head-injured group, and 23.26 +/- 1.53 ng/ml in the negative control group. Serum levels of S-100b seem to be a highly sensitive but not very specific marker for isolated neurotrauma. Measurement of this parameter may be helpful as an additional screening tool to identify high risk groups in the cohort of MHT patients.

Endotoxemia and cytokine generation in cardiac surgery in relation to flow mode and duration of cardiopulmonary bypass.

We investigated whether pulsatile flow in cardiopulmonary bypass (CPB), which has been shown to improve intestinal perfusion, reduces endotoxin translocation from the gut and, in consequence, decreases cytokine generation. The study population consisted of 48 adult patients who underwent elective CPB surgery. Pulsatile flow was used during aortic cross-clamping in 24 patients and nonpulsatile flow in 24 patients. Plasma endotoxin concentration increased in all patients during CPB. Significantly (P < 0.05) lower peak levels of 8.25 +/- 1.17 (SEM) pg/mL were reached 30 min after CPB in patients with pulsatile flow in contrast to 11.26 +/- 1.42 pg/mL in patients with nonpulsatile flow. The extent of endotoxemia was not related to the duration of CPB. Following the increase of plasma endotoxin, the concentrations of IL-6 and IL-8 increased with delay of approximately 1 h. The peak levels of these cytokines corresponded significantly (P < 0.005 and P < 0.01, respectively) with duration of CPB, but not with flow mode. Thus, in patients with CPB of more than 97 min (median), IL-6 reached a peak of 335.5 +/- 48.87 pg/mL and IL-8 of 64.86 +/- 24.79 pg/mL in contrast to 210.9 +/- 18.45 pg/mL and 21.2 +/- 10.19 pg/mL, respectively, with bypass times of less than 97 min. The degree of endotoxemia in CPB mainly depends on the quality of tissue perfusion. Cytokine generation, however, is not triggered exclusively by endotoxin, but rather by the trauma of CPB and surgery.

Bradykinin B2-receptor antagonism attenuates fatal cardiocirculatory breakdown induced by severe experimental pancreatitis.

OBJECTIVES: To investigate the impact of the long-acting bradykinin B2 receptor antagonist HOE 140 (Icatibant) on survival time in a model of severe porcine pancreatitis. DESIGN: Randomized, controlled intervention trial. SUBJECTS: Thirty domestic pigs of either gender anesthetized by intravenous application of piritramide, midazolam, and pancuronium and mechanically ventilated. INTERVENTIONS: Pancreatitis was induced by an injection of sodium taurocholate (5%, 1 mL/kg body weight [BW]) and enterokinase (10 U/kg BW). Control animals (group 1, n = 10) underwent the spontaneous course of the disease. In two treatment groups, Icatibant was administered either in a low (100 nmol/kg BW; group 2, n = 10) or in a high dosage (5000 nmol/kg BW; group 3, n = 10). MEASUREMENTS AND MAIN RESULTS: Mean survival time was significantly prolonged by Icatibant (controls, 6.6 hrs; group 2, 9.8 hrs; p = .022; group 3, 10.9 hrs; p = .007). Six hours postinduction, the decline of total peripheral resistance (52% of baseline) and cardiac index (92% of baseline) in controls was significantly improved by Icatibant, both in the low (16% and 44%; p < .05) and high (6% and 45%; p < .05) dosage. The concentrations of free, nonreceptor-bound kinin in plasma 6 hrs postinduction were significantly lower in controls than in groups 2 and 3 animals (111+/-50 vs. 208+/-40 and 237+/-52 fmol/mL, respectively). Six hours postinduction, the pretreatment with Icatibant was associated with significantly higher plasma concentrations of phospholipase A2 (controls, +1194%; group 2, +2000%; group 3, +2285% of baseline values) and interleukin-1 receptor antagonist (controls, 1900+/-800; group 2, 3100+/-800; group 3, 3600+/-800 pg/mL). In contrast, the increase of urinary trypsinogen activation peptides indicating local pancreatic damage (589+/-114 nmol/L in controls) was substantially attenuated by pretreatment with Icatibant (group 2, 467+/-102, NS; 352+/-91 nmol/L in group 3; p = .022 vs. controls). Systemic inflammatory reactions, however, as quantified by C-reactive protein and the extracellularly discharged neutrophil cytosolic inhibitor leukocyte neutral proteinase inhibitor were not influenced by the bradykinin B2-receptor antagonist. CONCLUSIONS: Pretreatment with the bradykinin B2 receptor antagonist Icatibant resulted in prolonged survival time and in delayed impairment of major macrocirculatory and pulmonary variables. Icatibant resulted in elevated concentrations of free, circulating kinin. This was associated with increased concentrations of phospholipase A2 and interleukin-1 receptor antagonist, suggesting that circulating kinins strengthen the activation of some mediator cascades, the association of which with the kinin metabolism requires further experimental clarification. Other variables indicating a systemic inflammatory response (C-reactive protein, leukocyte neutral proteinase inhibitor) remained unaffected by Icatibant. Bradykinin antagonism distinctly ameliorated the local pancreatic damage, indicated by increased urinary concentrations of trypsinogen activation peptides. It is concluded that the kinin metabolism plays an important role in the pathophysiology of systemic complications after severe experimental pancreatitis.

S-100b as a screening marker of the severity of minor head trauma (MHT)--a pilot study.

Due to its neural tissue specificity S-100b is considered as a screening marker of cerebral injury in head trauma patients. However, the occurrence and relevance of an increased S-100b serum level in minor head trauma (MHT) is still debated. Therefore, the purpose of our study was to evaluate the diagnostic utility of S-100b measurements in a level I trauma center emergency room (ER). Eighty patients presenting with clinical symptoms of MHT (GCS score of 13-15, transitory loss of consciousness, amnesia, nausea) were prospectively recruited. Blood samples were drawn at 0 h, 6 h and 24 h after admission, and a cerebral computed tomography (CT) was performed. The reference group consisted of 10 patients with severe head injury (GCS score < 8), the control group of 20 healthy volunteers. Concentrations of S-100b in serum were determined by an immunoluminometric assay. The results were compared with the plasma levels of polymorphonuclear (PMN) elastase as an established general trauma marker. In the MHT group, the S-100b serum level revealed 1.26 +/- 0.57 ng/ml at study entry (73.46 +/- 47.53 min after trauma). In comparison, the S-100b concentration was significantly elevated in patients with severe head trauma (5.26 +/- 1.65 ng/ml, p = 0.009), but no significant difference became evident in relation to the control group (0.05 +/- 0.01 ng/ml). Starting values of PMN elastase in plasma amounted to 66.40 +/- 14.92 ng/ml in severe trauma, and to 60.52 +/- 10.75 ng/ml in MHT showing significant differences only in relation to the control group (23.36 +/- 1.53 ng/ml). When correlated with the severity of the later clinical course, the first S-100b measurements exhibited steadily increasing values as demonstrated in MHT outpatients (0.29 +/- 0.11 ng/ml), MHT in-hospital patients (0.70 +/- 0.19 ng/ml) and MHT intensive care unit patients (5.03 +/- 3.18 ng/ml). PMN elastase levels revealed no significant differences concerning the three MHT subgroups. Thus, in contrast to the general trauma marker PMN elastase, assessment of the specific neuroprotein S-100b early after traumatic insult appears to be a promising laboratory marker for the prognosis of the severity of brain injury in MHT patients. Nevertheless, further investigations are required to better understand its predictive value.

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.

Proteolytic destruction of functional proteins by phagocytes in human peritonitis.

BACKGROUND: Besides phagocyte-derived oxidative autoaggression, proteolytic destruction of functional proteins in the peritoneal cavity may also be involved in the pathomechanism of secondary peritonitis. To evaluate the pattern of proteolysis, 43 patients undergoing initial operation for acute peritonitis (n = 30) or scheduled abdominal lavages (Etappenlavage) for resolution of persistent peritonitis (n = 13) and 16 surgical patients with abdominal exudation without peritonitis were enrolled in our study. MATERIALS AND METHODS: Thirty blood samples and purulent exudates were taken simultaneously in each peritonitis group at the surgical interventions. Sixteen clear exudates were obtained from patients with post-operative non-infectious irritations. The following parameters were measured: (a) elastase (from neutrophils) and cathepsin B (from monocytes/macrophages); (b) alpha1-proteinase inhibitor (alpha1PI) and overall cysteine proteinase inhibitor capacity; and (c) opsonic activity and degradation products of fibrinogen, complement C3 and immunoglobulin IgG. RESULTS: Circulating levels of phagocyte proteinases and of alpha1PI were significantly elevated, whereas antigen concentrations and opsonic activity of C3 and IgG were slightly reduced in peritonitis patients compared to healthy volunteers. No degradation products were detectable in patients' blood. Discharge of phagocyte proteinases was even more pronounced in both types of peritonitis exudates. Although most of the elastase was complexed with alpha1PI, active elastase and its specific fibrinogen split product was found along with significantly reduced inhibitory capacity for elastase and cysteine proteinases. Local opsonic activity was dramatically diminished because of proteolytic degradation of C3 and IgG. Despite some phagocyte proteinase release, no destruction of functional proteins was seen in clear exudates. CONCLUSIONS: Higher values of extracellularly released phagocyte proteinases concomitant with lower opsonin activity in exudates from patients with persistent peritonitis can be taken as a further hint of the involvement of local proteolysis-induced pathomechanisms in the development of lethal multiple organ failure, which occurred more frequently in patients with persistent peritonitis (54%) than in those with acute peritonitis (27%).

High-dose antithrombin III treatment of severely injured patients: results of a prospective study.

BACKGROUND: Antithrombin III (AT III) treatment has been shown to reduce disseminated intravascular coagulation and to inhibit thrombin, which plays a central role in the activation of platelets and other inflammatory systems in conditions with severe inflammation. The objective of this study was to evaluate the influence of early and high-dose administration of AT III to patients with severe multiple injuries on the inflammatory response and outcome. METHODS: In a placebo-controlled, double-blind study, 40 consecutive patients with Injury Severity Scores of 29 or greater who met the inclusion criteria were randomized to receive either AT III or placebo within 360 minutes after trauma. Twenty patients were administered AT III for a period of 4 days, aiming to achieve AT III concentrations of 140% of normal. RESULTS: The AT III and placebo groups were comparable with respect to Injury Severity Score, age, incidence of blood pressure less than 80 mm Hg on admission, initial base deficit, and start of the test drug. The patients in the AT III group received a total of about 20,000 IU during the first 4 days. AT III levels of 130 to 140% could be achieved by this regimen, whereas in the control group the AT III concentration averaged about 70%. In the AT III group prothrombin tended to be elevated and prothrombin fragment F1+2 as well as thrombin-AT III complex tended to be lower on the first day. No differences between groups, however, could be observed with respect to partial thromboplastin time, prothrombin time, platelets, plasminogen activator inhibitor I, soluble tumor necrosis factor receptor II, neutrophil elastase, interleukin (IL)-1 receptor antagonist, IL-6, and IL-8. Mortality (15 vs. 5%), incidence of respiratory failure (55 vs. 55%), duration of mechanical ventilation (13 vs. 12 days), and length of stay in the surgical intensive care unit (19 vs. 21 days) were also similar in both treatment groups. The duration of organ failure, however, was shorter in the patients receiving AT III. CONCLUSION: The early and high-dose administration of AT III to patients with severe blunt trauma appears not to attenuate the posttraumatic inflammatory response or to significantly improve outcome.

Local and systemic reactions after lung contusion: an experimental study in the pig.

The present study was designed to investigate the consequences of isolated unilateral lung contusion on local alveolar and systemic inflammatory responses in an animal model in the pig. Isolated unilateral lung contusion was induced by bolt shot in eight mechanically ventilated animals under general anesthesia (sham: n=4). Plasma and bronchoalveolar lavage fluid were collected during a period of 8 h following lung contusion. Leukocytes, leukocyte neutral protease inhibitor (LNPI), terminal complement complex (TCC), thrombin-antithrombin-complex (TAT) as well as pulmonary microvascular permeability and surfactant function were determined. Within 30 min, lung contusion was found to cause a significant local and systemic increase in TCC and TAT concentrations and a systemic increase in LNPI concentrations. The latter was accompanied by a sequestration of leukocytes in the contused lung. Complement activation and leukocyte sequestration in the contused lung progressively increased during the investigation period. Although surfactant function decreased in the entire lung 30 min after contusion, TCC, TAT, and leukocyte sequestration was unchanged in the contralateral lung. The first indication of an involvement of the contralateral lung was obtained by an increase in leukocyte sequestration 8 h after lung contusion. Unilateral lung contusion initiates an early systemic activation of humoral and cellular defense systems. Involvement of the contralateral lung appears to be a secondary event caused by a systemic inflammatory reaction.