Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein

BACKGROUND: Neutrophil apoptosis is crucial in the resolution of inflammation. The role of interleukin (IL)-8 in neutrophil apoptosis has not been previously studied; we hypothesized that in addition to its role as a chemoattractant, IL-8 would regulate polymorphonuclear leukocyte (PMN) apoptosis. METHODS: PMNs were adhered to plastic during hypoxia or normoxia and treated with IL-8 dosages of 0 to 1000 ng/mL. Apoptosis was assessed by cellular histology and the TUNEL assay. For receptor inhibition, blocking antibodies to IL-8 receptors in the presence of IL-8 were added. Apoptosis of PMNs treated with anti-Fas antibody +/- IL-8 was also analyzed. RESULTS: After treatment with 100 ng/mL IL-8 apoptosis was decreased from an average of 39.1% 9.3%. Inhibition of IL-8RA was able to restore apoptosis to 59.4%. Western analysis showed that with IL-8, there was a marginal decrease of total Fas protein, whereas Fas ligand was increased. After incubation with an apoptosis inducing-Fas antibody plus IL-8 reduced apoptosis to 9.5%. CONCLUSIONS: IL-8 not only promotes the inflammatory response by recruiting PMNs but also acts to suppress apoptosis mainly through the IL-8RA in an oxygen tension independent manner. The reduction in apoptosis is associated with changes in Fas and FasL where the presence of IL-8 suppresses the proapoptotic function of Fas-FasL interactions.     

Thyroid function in a population with an extra iodine intake]

Polymorphonuclear leukocyte (PMN) superoxide (.O2-) production has been implicated in the pathogenesis of cardiopulmonary bypass (CPB)-related end organ injury. PMN "priming" has been described as an event which enhances the release of .O2- following a second, activating insult. We hypothesized that PMN priming occurs during CBP and is temporally related to the plasma level of complement (C3a), interleukin (IL)-6, and IL-8. PMNs were isolated from 10 CPB patients pre-bypass (preCPB), 5 min after protamine administration (PROT), and at 6 and 24 h post-CPB. PMN .O2- production was measured by a cytochrome c reduction assay in the presence or absence of either phorbol 12-myristate-13-acetate (PMA, 0.4 microgram/ml) or N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 microM) and also after priming with 2000 nM platelet-activating factor (PAF) followed by activation with either PMA or FMLP. Plasma levels of C3a, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay. PMA-activated PMN .O2- production was significantly elevated at 6 h post-CPB compared to pre-CPB levels (11.04 +/- 0.9 vs 7.62 +/- 0.57, P = 0.009), indicating that CPB is associated with in vivo PMN priming. When PMNs were primed in vitro with PAF and then activated with PMA or FMLP, .O2- release at 6 h post-CPB was also significantly greater than pre-CPB levels (16.04 +/- 0.74 vs 12.2 +/- 0.92, P = 0.038; and 17.33 +/- 1.38 vs 13.33 +/- 1.35, P < 0.05), indicating that CPB acts synergistically with PAF to prime PMNs. Levels of C3a rose significantly over pre-CPB levels at PROT (P = 0.001), and IL-6 and IL-8 rose over pre-CPB levels at 6 h post-CPB (P = 0.01 and P = 0.006, respectively). These findings demonstrate that CPB not only directly primes PMNs, but also potentiates priming of PMNs by PAF. This "primed" PMN state, which coincided with the increased plasma levels of inflammatory mediators, may suggest a mechanism of predisposition to organ dysfunction following CPB.     

Leukocyte adhesion molecules in the liver and plasma cytokine levels in endotoxin-induced rat liver injury

BACKGROUND: The interactions between polymorphonuclear neutrophils (PMNs) and sinusoidal endothelial cells (SECs) have been known to be involved in the pathogenesis of acute liver injury. It has been also reported that tumor necrosis factor-alpha (TNF-alpha) up-regulates ICAM-1 expression on SECs and that interleukin-8 (IL-8) provokes rapid activation of CD11/CD18 on PMNs. These findings expand into the relationship between the expression of leukocyte adhesion molecules (ICAM-1, CD11a/CD18 and CD11b/CD18) in liver tissues and plasma TNF and IL-8 levels after lipopolysaccharide (LPS)-induced liver injury in rats. METHODS: Male Wistar rats weighing 200-250 g were treated with 2 mg LPS/kg intravenously in a 0.2- to 0.25-ml volume. Liver and blood samples were obtained at 1, 3, 8, and 12 h after LPS exposure. Plasma TNF and IL-8 levels were measured using bioassay and specific enzyme-linked immunosorbent assay, respectively. Liver samples were fixed and studied by immunohistochemistry using specific monoclonal antibodies against ICAM-1, CD11a, and CD11b. RESULTS: The TNF level showed a peak at 1 h (23.3 +/- 11.4 IU/ml), and the IL-8 level showed a peak at 3 h (343.1 +/- 110.5 ng/ml) after LPS exposure. An increase in the number of PMNs in the liver was observed as early as 1 h and continued until 12 h after LPS exposure. PMNs adhered to degenerated SECs and hepatocytes. ICAM-1 on SECs was diffusely and strongly expressed at 8 h, and PMNs adhered to SECs expressed both CD11a and CD11b. ICAM-1 was also observed on hepatocytes. CONCLUSION: These data suggest that PMN-SEC and PMN-hepatocyte interactions via leukocyte adhesion molecules, related to inflammatory cytokines such as TNF and IL-8, exist and play an important role in the pathogenesis of acute liver injury.     

Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation

Expression of mRNA for the neutrophil (PMN) chemokine, KC, in rat models of lung injury suggests a role for this chemokine in pulmonary inflammation. We addressed this hypothesis at the protein level by functionally characterizing recombinant rat KC (rKC) in vitro and in vivo. In vitro, rKC induced PMN chemotaxis and increased the expression of CD11b/CD18 on PMNs. Recombinant KC also induced a respiratory burst (quantitated by flow cytometry) in rat PMNs, similar to that caused by its human structural homologue, gro/melanoma growth-stimulating activity, on human PMNs, but less than that caused by IL-8 on human PMNs. Intratracheal instillation of rKC induced dose-dependent PMN influx into airspaces (average PMNs in bronchoalveolar lavage: vehicle = 1.5%, n = 4; rKC (1 microgram) = 11.5%, n = 2; rKC (10 micrograms) = 77.3%, n = 2). A neutralizing anti-KC Ab reduced the chemotactic activity of rat bronchoalveolar lavage fluid collected after the intratracheal administration of LPS (48.3 +/- 8% of control, n = 4). Anti-KC neutralizing Ab markedly inhibited PMN accumulation (71 +/- 6%) within the lungs in response to an intratracheal challenge of LPS. We conclude that rat KC is a major but not exclusive mediator of PMN activation and recruitment during LPS-induced pulmonary inflammation.     

Changes in plasma erythropoietin and interleukin-6 concentrations in patients with septic shock after hemoperfusion with polymyxin B-immobilized fiber

OBJECTIVE: To find out whether polymyxin B-immobilized fiber (PMX-F) treatment affects the clinical parameters and plasma concentrations of erythropoietin (EPO) and interleukin (IL)-6. DESIGN: A prospective case series study. SETTING: Intensive care unit of the Department of Internal Medicine, Misato Junshin Hospital, Saitama, and Koto Hospital, Tokyo, Japan. PATIENTS: 17 consecutive patients (10 men, 7 women; mean age 54.6 years) with clinically defined septic shock and 20 healthy volunteers (12 men, 8 women; mean age 52.2 years). MAIN RESULTS: Of the 17 patients with septic shock, 9 (53 %) survived. The systolic blood pressure increased significantly from 78+/-6 to 106+/-8 mm Hg 2 h after PMX-F treatment in patients with septic shock. Plasma endotoxin levels decreased significantly after treatment, from 40+/-6 to 12+/-4 pg/ml. The pretreatment plasma concentrations of EPO and IL-6 were significantly higher in the 8 nonsurviving patients with septic shock (EPO: 400+/-36 mlU/ml; IL-6: 6260+/-1180 pg/ml) than in the 9 surviving patients (EPO: 120+/-22 mlU/ml; IL-6: 680+/-138 pg/ml) and the 20 control subjects (EPO, 12+/-6 mlU/ml; IL-6, 8+/-2 pg/ml). Plasma concentrations of EPO and IL-6 in patients with septic shock decreased significantly after PMX-F treatment (EPO, nonsurviving: 320+/-28 mlU/ml, p < 0.05; survivors: 26+/-8 mlU/ ml, p < 0.001; IL-6, nonsurviving: 3860+/-840 pg/ml, p < 0.01; survivors: 84+/-20 pg/ml, p < 0.001). CONCLUSIONS: Plasma concentrations of EPO and IL-6 may be prognostic indicators in patients with septic shock: PMX-F treatment may be effective in reducing the plasma concentrations of EPO and IL-6 in patients with septic shock.     

Endothelial cell activation by leukocyte microparticles

The ability of polymorphonuclear leukocytes (PMNs) to modulate endothelial cell (EC) activation was investigated. Adding PMNs to cultured HUVECs resulted in a release of IL-6 (888 +/- 71 pg/ml, a 35-fold increase over release by the two cell types alone) and IL-8 (45.2 +/- 14.5 ng/ml, a 6.4-fold over PMN release alone and a 173-fold increase over EC release alone). In contrast, the release of TNF-alpha, IL-1beta, and platelet-derived growth factor was not affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or beta2 integrins or a physical segregation of PMNs and ECs did not reduce EC stimulation. In contrast, cell-free supernatants of PMNs recapitulated EC activation with an 18-fold up-regulation of EC IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with metabolic antagonists or membrane cross-linking agents all suppressed EC activation. By flow cytometry, PMNs released in the supernatant, heterogeneous membrane-derived microparticles containing discrete proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN microparticle formation was enhanced by inflammatory stimuli, including formyl peptide and phorbol ester, and was time-dependent, reaching a plateau after a 1-h incubation from stimulation. Purified PMN microparticles induced EC IL-6 release in a reaction that was quantitatively indistinguishable from that observed with unfractionated PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R. These findings demonstrate that membrane microparticles released from stimulated PMNs are competent inflammatory mediators to produce EC activation and cytokine gene induction.     

The role of neutrophils in peritoneal adhesion formation

The most common cause of intraperitoneal adhesions is previous abdominal surgery. Postoperative adhesion formation results from a fibroproliferative inflammatory reaction that begins with an influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity. Adherence of the PMNs to the endothelial cells (EC) is necessary for PMN migration into the tissue in response to a stimulus. Several receptor-counterreceptor pairs of ligands such as CD11/CD18 on the PMN and ICAM-1 (CD54) on EC have been identified. Monoclonal antibody against CD11/CD18 (R15.7) inhibits PMN adherence and migration and consequently protects against PMN-induced tissue injuries. We therefore studied the effect of preventing PMN-EC adherence, using anti-CD18 monoclonal antibody, on postoperative adhesion formation in rabbits. Group 1 was a control receiving physiologic saline, and group 2 received anti-CD18 antibody (R15.7, 2 mg/kg). The treatment was administered iv at the end of surgery and repeated on the first and second postoperative days. Peritoneal adhesions were induced at laparotomy by repairing two peritoneal defects, by oversewing the defect (model 1), and by resuturing the removed parietal peritoneum in its place as an ischemic graft (model 2). Adhesions were evaluated blindly at 10 days after operation by measuring the percentage of the suture line covered with adhesions (model 1) or by a scoring system (model 2). All control animals developed intraperitoneal adhesions and the percentage of the suture line covered with adhesions was 25 +/- 5.9% (mean +/- SEM) and the mean score in model 2 was 0.9 +/- 0.2. Anti-CD18 antibody, R15.7, increased the degree of postoperative adhesion formation in both models, but the results were significant only in model 2. Also, anti-CD18 antibody significantly decreased peritoneal neutrophils from 11.1 x 10(7) +/- 1.8 x 10(7) to 2.2 x 10(7) +/- 0.4 x 10(7) (P < 0.001) on the first postoperative day. It is concluded that inhibition of PMN-EC adherence does influence the postoperative adhesion formation. These results might suggest that PMNs have a role in modulating postoperative adhesion formation.     

High levels of interleukin-8 in the blood and alveolar spaces of patients with pneumonia and adult respiratory distress syndrome

There is ample experimental evidence that polymorphonuclear neutrophils (PMN) play a critical role in the pathogenesis of the adult respiratory distress syndrome (ARDS). Since interleukin-8 (IL-8) is a strong chemotactic factor for PMN, we measured IL-8 levels in plasma and bronchoalveolar lavage (BAL) fluid of 18 patients, 12 with ARDS and 6 with severe pneumonia uncomplicated by ARDS, all of whom had an increased number of PMN in BAL fluid. Seven healthy subjects served as controls. We found elevated levels of IL-8 in the alveolar spaces of all patients tested. Elevated BAL IL-8 levels were related to a fatal outcome and the presence of shock and correlated with a general clinical severity index (simplified acute physiological score). BAL fluid levels of IL-8 were significantly higher in patients with ARDS than in patients with pneumonia. In plasma, IL-8 levels were increased similarly in all patients and did not correlate with survival or the presence of shock. The BAL fluid-to-plasma ratio of IL-8 was significantly greater than that of tumor necrosis factor alpha, indicating higher local production of IL-8. Moreover, the presence of a primed subpopulation of blood PMN with respect to H2O2 production indicates that IL-8 may contribute to the neutrophil-mediated process in the pathogenesis of ARDS and pneumonia.     

Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model)

Carbohydrate moieties on leukocytes adhere to activated platelets via P-selectin under static binding condition studies. We characterize polymorphonuclear cell (PMN) surface interactions with surface adherent platelets and the PMNs response, under physiologic flow conditions corresponding to a shear of 100 s-1, in an in vitro flow chamber. Fluorescent labeled PMNs with red blood cells were drawn through a transparent flow channel and visually quantitated over 30 minutes, interacting with a confluent monolayer of activated, shear-spread platelets expressing P-selectin. PMN adhesion was saturable (2,250 +/- 350/mm2), and time and cation (Ca2+, Mg2+) dependent, and PMNs did not bind to the experimental surface in the absence of a platelet monolayer. P-selectin antibodies completely abolished PMN adhesion in a concentration-dependent manner with half inhibition at 70 micrograms/mL. Antibodies to a putative P-selectin receptor CD15 (80H5 and MMA) maximally inhibited PMN adhesion by 73% and 10%, respectively. Adherent PMNs appeared morphologically activated and flow cytometric analysis of adherent PMNs confirmed activation because CD11b and CD18 surface expression was upregulated (100% and 27%, respectively), whereas L-selectin was downregulated (55%) compared with control nonadherent PMNs. In the presence of the metabolic inhibitor sodium azide (0.02% and 0.1%) there was a 23% +/- 9% and 51% +/- 3% decrease, respectively, in PMN adhesion at 100 s-1. Thus, P-selectin is required for PMN adhesion to a pathophysiologic surface of activated adherent platelets at physiologic shear rates. Furthermore, a secondary step involving PMN activation after platelet binding appears necessary for complete (irreversible) adhesion to occur. This unique flow cell provides a model to explore, under controlled conditions, biologic mechanisms and ligands involved in leukocyte-platelet binding that play important roles in PMN localization at sites of thrombosis and vascular injury.     

Extracellular matrix regulates apoptosis in human neutrophils

BACKGROUND: During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. METHODS: Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. RESULTS: PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. CONCLUSIONS: ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.     

Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS

BACKGROUND: Oxidant stress is a purported mechanism of tissue damage in patients with ARDS and acute lung injury (ALI). Isoprostanes, prostanoid compounds primarily formed nonenzymatically via lipid peroxidation, are precise markers of in vivo oxidant stress. Plasma levels of metabolites of 8-iso-prostaglandin-F2alpha (8-iso-PGF2alpha) correlate with outcome in patients with ARDS. OBJECTIVE: To examine exhaled breath condensate levels of 8-iso-PGF2alpha as a noninvasive quantification of pulmonary oxidant stress in patients with, or at risk for, ARDS/ALI. METHODS: Breath condensate was collected from 22 patients with, or at risk for, ARDS/ALI by placing Tygon tubing submerged in an ice bath in line with the expiratory limb of the ventilator circuit. Ten patients without lung disease, who were intubated while undergoing minor surgical procedures, served as control subjects. Between 1 and 3 mL of condensate was collected over a 30- to 60-min period, then immediately frozen and stored at -70 degrees C until analysis. The 8-iso-PGF2alpha was purified and derivatized, then quantified by stable isotope dilution in conjunction with gas chromatography/mass spectrometry. RESULTS: The mean level of exhaled 8-iso-PGF2alpha in the patients with ALI/ARDS, 87+/-28 pg/mL, was significantly higher than the mean in the normal group, 7+/-4 pg/mL (p = 0.007). The 8-iso-PGF2alpha levels were greater than two standard deviations above the mean of the normal group in 12 of 22 patients with or at risk for ARDS/ALI. CONCLUSIONS: These results provide further evidence that lipid peroxidation does occur in patients with ARDS/ALI. The measurement of exhaled isoprostanes provides a novel, noninvasive method to quantify oxidant stress in such patients.     

Elevated serum cytokines are associated with cytomegalovirus infection and disease in bone marrow transplant recipients

To assess the relationship between serum cytokines and cytomegalovirus (CMV) reactivation, 75 allogeneic bone marrow transplant patients underwent weekly measurements of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-alpha, CMV blood cultures, and antigenemia tests. Of the patients, 44 (58.7%) developed CMV infection, and 19 (25.3%) developed clinical CMV disease. The mean maximum levels of all three cytokines were significantly increased in patients with CMV infection compared with levels in those without. Maximum levels of IL-6 were significantly higher in patients with active CMV disease than in those who did not develop CMV disease (281.2+/-85.5 vs. 95.7+/-15.0 pg/mL; P=.034). Levels of IL-8 and TNF-alpha were also elevated in patients who developed active disease. In a multivariate logistic regression model, IL-6 levels were independently associated with CMV disease (odds ratio=1.70 per 100-pg/mL increase in IL-6; P=.009). Cytokines may play an important role in the pathogenesis of CMV after bone marrow transplantation and may be a useful predictor for CMV.     

Interleukin 8 released after acute myocardial infarction is mainly bound to erythrocytes

OBJECTIVE: To determine whether rapid clearance of interleukin 8 (IL-8) from plasma through binding to the erythrocyte chemokine receptor may be responsible for failure to detect IL-8 consistently after acute myocardial infarction. DESIGN: Plasma concentrations of IL-8 were measured at frequent intervals in 43 consecutive patients. In 21 of these, erythrocyte bound IL-8 concentrations were also measured. The influence of infarct size, type of treatment, and the presence of early successful reperfusion on IL-8 release was assessed. RESULTS: Peak IL-8 concentrations in plasma were raised in 31 of the 43 patients (68%). Median plasma IL-8 concentrations were 16.0 pg/ml (range 2.4 to 225.0 pg/ml) six hours after the onset of chest pain. Twelve hours after the onset of symptoms, plasma IL-8 concentrations had already returned to normal in 27 patients. In contrast, in 18 of 21 patients (86%), erythrocyte bound IL-8 concentrations were raised at between 6 and 30 hours, with a median peak value of 59.8 pg/ml (range 19 to 148 pg/ml). No correlation between peak creatine kinase MB and peak IL-8 (plasma or erythrocyte bound) was observed. There was a significant difference in peak plasma IL-8 concentrations between patients who underwent direct PTCA (19.4 pg/ml) and those who received conservative treatment (9.9 pg/ml; p = 0.0206), but no correlation with the presence of early successful reperfusion. CONCLUSIONS: IL-8 is released in plasma after acute myocardial infarction and subsequently binds to red blood cells, resulting in only a transient rise of plasma IL-8 and a more prolonged increase of erythrocyte bound IL-8.     

Endogenous interleukin-8 (IL-8) surge in granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization

The relationship between stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) and the endogenous production of interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) was studied in normal donors for allogeneic peripheral blood stem cell (PBSC) transplantation. G-CSF was administered to 20 normal donors at a dose of 10 microg/kg/d for 5 days with aphereses on days 5 and 6 of G-CSF treatment. Cytokine serum levels were measured using an enzyme-linked immunosorbent assay (ELISA) before and during G-CSF treatment. Before treatment, the average level of IL-8 was 7.1 pg/mL, increasing to 207.0 pg/mL on day 5 and 189.1 pg/mL on day 6. Serum IL-8 levels correlated CD34(+) cell numbers (P =.0151 and P =.0005 on days 5 and 6, respectively) and colony-forming unit-granulocyte-macrophage (CFU-GM) numbers (P =.0019 and P =.0010 on days 5 and 6, respectively). Furthermore, preapheresis serum IL-8 levels correlated with the yield of CD34(+) cells (P =.0027). In contrast, before treatment, the average levels of MIP-1alpha, TNF-alpha, and IFN-gamma were 70.1, 4.03, and 3.84 pg/mL, respectively, and no significant changes in the levels of these cytokines were observed during G-CSF treatment. These studies suggest that IL-8 production may be critical to G-CSF-induced stem cell mobilization, although the underlying mechanism could not be clarified.     

Stimulation of inflammatory markers after blunt trauma

BACKGROUND: Inflammatory mediators are released after trauma and may be related to the pathogenesis of sepsis. A prospective combined study of the pattern of release of an inflammatory mediator, interleukin (IL) 6, leucocyte activation (polymorphonuclear leucocyte (PMN) CD11b receptor expression and plasma elastase-alpha1 proteinase inhibitor complex (E-alpha1PI)) and soluble endothelial adhesion molecule expression (soluble E-selectin (sE-selectin) and soluble intracellular adhesion molecule 1 (sICAM-1)) was performed in patients suffering blunt trauma without adult respiratory distress syndrome (ARDS) or multiple organ failure syndrome (MOFS). METHODS: Thirty-one patients with a mean Injury Severity Score (ISS) of 14 (range 9-57) were studied. Venous blood samples were collected within 6 h of injury and then at 1, 3, 5 and 7 days. Leucocyte CD11b expression was quantified by flow cytometry. Serum IL-6, plasma E-alpha1PI, sE-selectin and sICAM-1 were measured by enzyme-linked immunosorbent assay. RESULTS: Serum IL-6, CD11b expression and E-alpha1PI levels were significantly raised above control values (P < 0.0001) on admission, slowly returning towards control values over the study period (median IL-6, 140 pg/ml versus undetectable; CD11b, 14.8 versus 6.4 mean channel fluorescence units; E-alpha1 PI, 208 versus 52 microg/l). The sICAM-1 level rose to a median of 539 ng/ml at 5 days (control 243 ng/ml). The median sE-selectin level also progressively increased to a maximum level of 80 ng/ml at 5 days (control 49 ng/ml). Eleven patients developed postoperative sepsis. Significant differences in CD11b expression were seen at days 3, 5 and 7 and in E-alpha1 PI at 6 h, 24 h and 3 days in patients who subsequently developed sepsis (P < 0.05). Severe injury (ISS 16 or greater) was associated with significantly greater responses in these measurements. CONCLUSION: These data show that markers of inflammation are specifically stimulated by trauma even when ARDS and MOFS do not occur. The CD11b receptor on PMNs may be useful in screening patients destined to develop post-traumatic sepsis.     

Serum levels of interleukin 1 and tumor necrosis factor alpha correlate with peritoneal adhesion grades in humans after major abdominal surgery

Peritoneal adhesions are a leading cause of potential morbidity and mortality. We undertook this prospective study to determine the clinical relevance of interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) levels as biological markers for peritoneal adhesion formation in humans. Fifteen patients who had previous colectomies and were undergoing re-exploration for an elective vascular procedure were studied. Blood samples were collected from each patient preoperatively and 30 minutes after the abdominal incision was made. Serum levels of IL-1 and TNF-alpha were determined using enzyme-linked immunosorbent assay kits. Adhesions were graded using an adhesion scale of 0 (none), 1 (mild), 2 (moderate), and 3 (extensive, dense). Preoperative levels of IL-1 and TNF-alpha did not differ significantly among all patients (IL-1 level was 60 +/- 14 pg/mL, and TNF-alpha level was 45 +/- 11 pg/mL; mean +/- standard deviation). Significant correlation was observed between grades of adhesions and early intraoperative levels of IL-1 [101 +/- 36 pg/mL for grade 1 (n = 8) vs 298 +/- 73 pg/mL for grade 3 (n = 6); P < 0.01] and TNF-alpha (88 +/- 23 pg/mL for grade 1 vs 261 +/- 88 mL for grade 3; P < 0.02). We conclude that early elevations of IL-1 and TNF-alpha are reliable biological markers for postoperative adhesions in humans. Studies utilizing cytokines antibodies to these markers may further elucidate the efficacy of this method for prevention of peritoneal adhesions.     

Soluble cytokine receptors and receptor antagonists are sequentially released after trauma

Cytokine receptors and receptor antagonists (RAs) have been identified in trauma patients. We hypothesized that after traumatic injury, a sequential release of soluble cytokine receptors and RAs may exist that mirrors the release of the primary cytokines themselves. Twenty-two patients were included in the study: 14 males and 8 females. The mean age was 30.1 +/- 12.5 (range, 19 to 71), and the mean Injury Severity Score was 28.7 +/- 12.6 (range, 4 to 57). There were 15 survivors and 7 nonsurvivors. Samples were collected on arrival to the emergency department and at serial intervals for up to 7 days. Monoclonal antibody enzyme-linked immunosorbent assay kits to tumor necrosis factor (TNF), soluble TNF-receptor (sTNF-R) 55 kd and 75 kd, interleukin (IL)-1 and IL-1 RA, and IL-2 and IL-2r were used. Sera from 22 healthy individuals were used as normal controls. No TNF, IL-1, or IL-2 could be detected in any patient sera after injury. Control levels for the soluble cytokine receptors and RAs were as follows: sTNF-R 55 kd, 607 +/- 89 pg/mL; sTNF-R 75 kd, 2,141 +/- 169 pg/mL; IL-1 RA, 291 +/- 35 pg/mL; and IL-2r, 426 +/- 53 U/mL. In trauma patients, both 55 kd and 75 kd sTNF-R were significantly elevated on arrival to the emergency department, with values of 2,441 +/- 506 pg/mL (p < 0.001) and 4,736 +/- 537 pg/mL (p < 0.001), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of polymorphonuclear leukocytes in collar-induced intimal thickening in the rabbit carotid artery

In this study, the involvement of polymorphonuclear leukocytes (PMNs) in the development of intimal thickening was investigated. A fibromuscular intima was induced by placing a silicone collar around the rabbit carotid artery for 3 days or 2 weeks; the contralateral artery was sham operated. Rabbits received placebo treatments (groups 1 and 3), granulocyte-colony stimulating factor (group 2; G-CSF, 20 microg x kg(-1) x d(-1), delivered by subcutaneous osmotic pumps), or an anti-CD18 monoclonal antibody (group 4; 1.5 mg/kg i.v.). The G-CSF treatment raised the peripheral PMN count 5- to 12-fold but had no effect on intimal thickening on day 3, 12, or 14. A single injection of anti-CD18 prevented PMN extravasation 6 hours after collar implantation without influencing intimal hyperplasia on day 14. Repeated daily administration of anti-CD18 strongly bound to CD18 on peripheral PMNs and inhibited both PMN-dependent plasma extravasation in the skin and accumulation of CD14-immunoreactive leukocytes in the intima and media. However, anti-CD18 did not suppress early intimal thickening or accumulation of alpha-smooth muscle actin-immunoreactive cells by day 3. It thus appears that the PMN influx in the intima and media evoked by the perivascular collar is of little functional relevance to the subsequent smooth muscle cell migration and intimal thickening in this model.     

Cytokine production regulating Th1 and Th2 cytokines in hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is caused by the hyperactivation of T cells and macrophages. The clinical characteristics associated with this disease result from overproduction of Th1 cytokines including interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-alpha). In this study, we analyzed the production of IL-12 and IL-4, which determine Th1 and Th2 response, respectively, and IL-10, which antagonizes Th1 cytokines, in 11 patients with HLH. IL-12 was detected in plasma in all patients (mean peak value, 30.0 +/- 5.0 pg/mL), while IFN-gamma was massively produced in nine patients (mean peak value, 79.2 +/- 112.0 U/mL). IL-4 was not detected in any of the patients. Plasma IL-10 levels were elevated in all patients (mean peak value, 2,698.0 +/- 3,535.0 pg/mL). There was a positive correlation between the levels of IFN-gamma and IL-10 (P < .01). The plasma concentrations of these cytokines were initially high, before decreasing after the acute phase. However, the decrease in IL-10 levels was slower than that of IFN-gamma. Although the concentration of IL-12 was high at the acute phase, in some patients, a peak in the level was delayed until the chronic phase. Thus, in HLH, production of cytokines that promote development of Th1 cells appears to be predominant over that for Th2 cell development. Overproduction of IL-10 was also observed indicating that a mechanism suppressing hyperactivation of Th1 cells and monocytes/macrophages functions in patients with this disease.     

Estrogen receptor alpha and beta expression in upper gastrointestinal tract with regulation of trefoil factor family 2 mRNA levels in ovariectomized rats

STUDY OBJECTIVE: To investigate the effect of short-term inhalation of nitric oxide (NO) on transpulmonary angiotensin II formation in patients with severe ARDS. DESIGN: Prospective, clinical study. SETTING: Anesthesiology ICU of a university hospital. PATIENTS: Ten ARDS patients who responded to inhalation of 100 ppm NO by decreasing their pulmonary vascular resistance (PVR) by at least 20 dyne x s x cm(-5) were included in the study. INTERVENTIONS AND MEASUREMENTS: In addition to standard treatment, the patients inhaled 0, 1, and 100 ppm NO in 20-min intervals. Fraction of inspired oxygen was 1.0. Hemodynamics were measured and recorded online. Mixed venous (pulmonary arterial catheter) and arterial (arterial catheter) blood samples were taken simultaneously for hormonal analyses at the end of each inhalation period. RESULTS: Pulmonary arterial pressure decreased from 33+/-2 mm Hg (0 ppm NO, mean+/-SEM) to 29+/-2 mm Hg (1 ppm NO, p<0.05), and to 27+/-2 mm Hg (100 ppm NO, p<0.05, vs 0 ppm). PVR decreased from 298+/-56 (0 ppm NO) to 243+/-45 dyne x s x cm(-5) (1 ppm NO, not significant [NS]), and to 197+/-34 dyne x s x cm(-5) (100 ppm NO, p<0.05, vs 0 ppm). Arterial oxygen pressure increased from 174+/-23 mm Hg (0 ppm NO) to 205+/-26 mm Hg (1 ppm NO, NS), and to 245+/-25 mm Hg (100 ppm NO, p <0.05, vs 0 ppm). Mean plasma angiotensin II concentrations were 85+/-20 (arterial) and 57+/-13 pg/mL (mixed venous) during 0 ppm NO and did not change during inhalation of 1 and 100 ppm NO. Mean transpulmonary plasma angiotensin II concentration gradient (=difference between arterial and mixed venous blood values) was 28+/-8 pg/mL (range, 0 to 69) during 0 ppm NO and did not change during inhalation of 1 and 100 ppm NO. Mean transpulmonary angiotensin II formation (transpulmonary angiotensin II gradient multiplied with the cardiac index) was 117+/-39 ng/min/m2 (range, 0 to 414) during 0 ppm NO and did not change during inhalation of 1 and 100 ppm NO. Mean arterial plasma cyclic guanosine monophosphate concentration was 11+/-2 pmol/mL (0 ppm NO), did not change during 1 ppm NO, and increased to 58+/-8 pmol/mL (100 ppm NO, p<0.05). Arterial plasma concentrations of aldosterone (142+/-47 pg/mL), atrial natriuretic peptide (114+/-34 pg/mL), angiotensin-converting enzyme (30+/-5 U/L), and plasma renin activity (94+/-26 ng/mL/h of angiotensin I) did not change. CONCLUSION: The decrease of PVR by short-term NO inhalation in ARDS patients was not accompanied by changes in transpulmonary angiotensin II formation. Our results do not support any relationship between transpulmonary angiotensin II formation and the decrease in PVR induced by inhaled NO.