Epinephrine inhibits tumor necrosis factor-alpha and potentiates interleukin 10 production during human endotoxemia

Short-term preexposure of mononuclear cells to epinephrine inhibits LPS-induced production of TNF, whereas preexposure for 24 h results in increased TNF production. To assess the effects of epinephrine infusions of varying duration on in vivo responses to LPS, the following experiments were performed: (a) Blood obtained from eight subjects at 4-24 h after the start of a 24-h infusion of epinephrine (30 ng/kg per min) produced less TNF after ex vivo stimulation with LPS compared with blood drawn before the start of the infusion, and (b) 17 healthy men who were receiving a continuous infusion of epinephrine (30 ng/kg per min) started either 3 h (EPI-3; n = 5) or 24 h (EPI-24; n = 6) were studied after intravenous injection of LPS (2 ng/kg, lot EC-5). EPI-3 inhibited LPS-induced in vivo TNF appearance and also increased IL-10 release (both P < 0.005 versus LPS), whereas EPI-24 only attenuated TNF secretion (P = 0.05). In separate in vitro experiments in whole blood, epinephrine increased LPS-induced IL-10 release by a combined effect on alpha and beta adrenergic receptors. Further, in LPS-stimulated blood, the increase on IL-10 levels caused by epinephrine only marginally contributed to concurrent inhibition of TNF production. Epinephrine, either endogenously produced or administered as a component of sepsis treatment, may have a net antiinflammatory effect on the cytokine network early in the course of systemic infection.     

Interleukin-10 controls interferon-gamma and tumor necrosis factor production during experimental endotoxemia

Interleukin-10 (IL-10) is a potent inhibitor of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and has been shown to protect mice from endotoxin shock. As IFN-gamma is another important mediator of LPS toxicity, we studied the effects of IL-10 on LPS-induced IFN-gamma synthesis in vitro and in vivo. First, we found that the addition of recombinant human IL-10 (rhIL-10) (10 U/ml) to human whole blood markedly suppressed LPS-induced IFN-gamma release while neutralization of endogenously synthesized IL-10 resulted in increased IFN-gamma levels. The ability of rIL-10 to inhibit LPS-induced IFN-gamma synthesis was also observed in vivo in mice. Indeed, administration of 1000 U recombinant mouse IL-10 (rmIL-10) 30 min before and 3 h after challenge of BALB/c mice with 100 micrograms LPS resulted in a threefold decrease in peak IFN-gamma serum levels. We then examined the production and the role of IL-10 during murine endotoxemia. We found that LPS injection causes the rapid release of IL-10, peak IL-10 serum levels being observed 90 min after LPS challenge. Neutralization of endogenously produced IL-10 by administration of 2 mg JES5-2A5 anti-IL-10 monoclonal antibody (mAb) 2 h before LPS challenge resulted in a marked increase in both TNF and IFN-gamma serum levels while irrelevant isotype-matched mAb had no effect. The enhanced production of inflammatory cytokines in anti-IL-10 mAb-treated mice was associated with a 60% lethality after injection of 500 micrograms LPS, while all mice pretreated with control mAb survived. We conclude that the rapid release of IL-10 during endotoxemia is a natural antiinflammatory response controlling cytokine production and LPS toxicity.     

Interleukin-6 stimulates coagulation, not fibrinolysis, in humans

The role of IL-6 as a mediator of haemostatic changes during severe inflammation is controversial. To assess the effect of IL-6 on haemostasis we conducted a controlled cross-over study in eight patients with metastatic renal cell cancer. In all subjects coagulation and fibrinolysis were monitored during and after a 4-h infusion of either 150 micrograms recombinant human (rh) IL-6, or during infusion of saline (control study). Mean maximum IL-6 concentrations were 1418.0 +/- 755.8 pg/ml. Compared to the control study, rhIL-6 induced activation of coagulation as reflected by a 190 +/- 55% increase in the plasma levels of thrombin-antithrombin III complexes (p < 0.001) and by a 24 +/- 11% increase in the plasma levels of in the prothrombin activation fragment F1 + 2 (p < 0.001). In contrast, fibrinolysis was not affected. We conclude that in severe inflammation IL-6 may contribute to the activation of coagulation, whereas other factors mediate changes in fibrinolysis.     

Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity

Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in Fc gamma IIR, Fc gamma IIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CDIIb (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.     

Intravascular coagulation and fibrinolysis

Intravascular coagulation occurs as a sequela of many diverse conditions and may vary greatly in clinical and laboratory manifestations. The essence of the problem is that plasma is converted to serum in the circulation. As a result, both hemorrhagic and thrombotic events may occur. Platelet count and fibrinogen determination are the most important diagnostic tests. If values are abnormal, tests for fibrin (fibrinogen) degradation products are indicated. The first step in management is to identify and attempt to eliminate the underlying cause. Heparin therapy should be considered, particularly when clotting and severe fibrinolysis are both present. Replacement of clotting factors may be considered, but its value is a matter of debate.     

Atherosclerosis: coagulation and fibrinolysis

Treatment of wild-type human immunodeficiency virus [HIV-1(IIIB)]-infected cell cultures with the thiocarboxanilide UC-781 under low selective pressure (i.e., 0.01 microg/ml) resulted in the emergence of V106A RT mutant virus. On increasing drug concentrations (stepwise up to 30 microg/ml) the virus retained the V106A RT mutation but acquired the novel F227L mutation in the RT genome in addition to the L100I, K1O1I, and Y181C mutations. This multiple-mutant virus proved highly resistant to virtually all nonnucleoside RT inhibitors (NNRTIs) (e.g., nevirapine, delavirdine, and loviride), but retained full sensitivity to nucleoside analogs such as AZT, ddI, (-)FTC, and 3TC. The F227 amino acid is highly conserved in HIV-1 strains and forms part of the NNRTI-binding pocket. Our model suggests a hydrophobic interaction between F227 and the chloro atom of UC-781.     

Interleukin-10 inhibits the in vitro proliferation of human activated leukemic CD5+ B-cells

Umbilical cord blood mononuclear cells isolated by density centrifugation are contaminated by erythrocytes and nucleated erythroid precursors which may exceed 50% of the total cell population, and thus interfere with phenotypic, functional and mRNA analyses. Lysis with hypotonic ammonium chloride can overcome this problem, but interferes with lysosomal function and should be avoided when cell preparations are intended for functional studies. The aim of this study was to develop a technique for removing erythroid cells from cord blood mononuclear cell preparations that would be as effective as ammonium chloride lysis but would not affect cellular function. This paper describes a method using 10F7, a mouse monoclonal antibody against human glycophorin A, and magnetic beads coated with anti-mouse immunoglobulin. The population of cord blood mononuclear cells recovered using this technique was of high purity, good yield and viability, and the cells responded appropriately to stimulation in vitro. To maximise cost-effectiveness, purification with magnetic beads could be performed after two density separations to reduce the quantity of beads required.     

Changes in the activation markers of blood coagulation and fibrinolysis in the neonatal period

BACKGROUND: Activation markers of the clotting and fibrinolytic systems are elevated immediately after birth and decline to near adult levels during the first 24 hours of life. The aims of this study were to investigate, whether the activation of both clotting and fibrinolysis is dependent on the mode of delivery, and to measure activation markers in newborns with infection beyond the first days of life. PATIENTS: We have studied activation markers thrombin-antithrombin III complex, prothrombin fragment 1 + 2, D-dimer and plasmin-antiplasmin complex by use of commercially available ELISA techniques in 20 newborns after elective Cesarean sections because of previous sections, in 20 newborns after Cesarean sections and a trial of labor with uterine contractions over a period of > 20 hours and in 20 newborns (34.-41. gestational week) aged 10-25 days with infection. 20 healthy adults served as controls. RESULTS: A significant elevation of all activation markers was observed both in the newborns after Cesarean sections and in the 10-25 days old children with infection. There were no differences among newborns after elective sections compared to newborns after section and a trial of labor with uterine contractions over a period of > 20 hours. CONCLUSIONS: The clotting and fibrinolytic systems reveal increased activation immediately after delivery, but uterine contractions over a period of > 20 hours seem not to make a difference. During infection, the activation markers of the hemostatic system in newborns aged 10-25 days behaves similarly to the mature adult system.     

Interleukin-10 gene transfer inhibits murine mammary tumors and elevates nitric oxide

Transfection of cDNA for IL-10 into line 66.1 murine mammary tumor cells results in marked suppression of tumor growth and metastasis. Others have reported that nitric oxide has potent antitumor activity and IL-10 is known to regulate the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We identified nitric oxide production in mammary tumors as indicated by electron paramagnetic resonance detection of nitric oxide-hemoglobin (NO-Hb). IL-10 expression resulted in elevated levels of NO-Hb in mammary tumors. Immunohistochemical examination of mammary tumors for iNOS protein revealed few positively staining cells in parental or control neo-transfected tumors but strong iNOS staining in all IL-10 transfected tumors, consistent with the NO-Hb data. To determine if mammary epithelial tumor cells themselves, express nitric oxide synthase activity, cultured tumor cells were treated with pro-inflammatory cytokines and nitrite accumulation was assessed in the conditioned medium. All IL-10 producing cell lines accumulated uM concentrations of nitrite in response to short term (24 hr) cytokine stimulation. Cells not expressing IL-10 (parental and neo-transfectants) accumulated no nitrite under similar culture conditions. After longer stimulation (48 hr), parental and 66-neo cells accumulated lower amounts of nitrite. IL-10 gene transfer is associated with increased iNOS protein expression and enzymatic activity detected both in vitro and in vivo. Our findings suggest that the antimetastatic and antitumor activity of IL-10 is related to enhanced production of nitric oxide.     

Interleukin-10 inhibits erythropoietin-independent growth of erythroid bursts in patients with polycythemia vera

In polycythemia vera (PV) erythroid colonies that grow in vitro in the absence of exogenous erythropoietin (EPO) arise from the abnormal clone that is responsible for overproduction of red blood cells. Although the mechanism of autonomous formation of burst-forming units-erythroid (BFU-E) is not fully understood, a spontaneous release of growth regulatory molecules by PV cells and/or by accessory cells is likely to be involved. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate abnormal erythropoiesis in PV. We studied the effect of recombinant human IL-10 on the EPO-independent growth of erythroid bursts derived from peripheral blood mononuclear cells (PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent, and specific inhibitory effect on autonomous BFU-E formation. Ten nanograms per milliliter of IL-10 significantly suppressed spontaneous growth of erythroid colonies in methylcellulose in five of five PV patients tested with a mean inhibition by 81% (range, 72-94). To elucidate the possible mechanism of the inhibitory action of IL-10 we further studied the effect of anticytokine antibodies on autonomous BFU-E growth and the ability of exogenous cytokines to restore IL-10-induced suppression of erythroid colony growth. Among a panel of growth regulatory factors tested (granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-3, granulocyte colony-stimulating factor, stem cell factor, and insulin-like growth factor-1) GM-CSF was the only molecule for which both an inhibition of spontaneous BFU-E formation by its respective antibody as well as a significant restimulation of erythroid colonies in IL-10-treated cultures by exogenous addition was found. Moreover, inhibition of GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level. Our data indicate that autonomous BFU-E growth in PV can be profoundly inhibited by IL-10 and that this inhibitory effect seems to be at least in part secondary to suppression of endogenous GM-CSF production.     

Activation of coagulation and fibrinolysis in open and laparoscopic cholecystectomy

BACKGROUND: Activation of coagulation and fibrinolysis occurs as a stress response to surgery and may predispose the patient to thromboembolic complications. Other components of the surgical stress response (cytokine release, neurohumoral response, etc.) have been shown to differ between laparoscopic and open cholecystectomy, and the aim of this study was to investigate the effects of laparoscopic and open surgery on the coagulation and fibrinolytic pathways. METHODS: Fourteen patients undergoing laparoscopic cholecystectomy and 12 patients undergoing open cholecystectomy had blood taken in the perioperative period for fibrinopeptide A (FPA) prothrombin fragment F1.2, antithrombin 3, tissue plasminogen activator (tPA) and its fast-acting inhibitor plasminogen activator inhibitor-1 (PAI-1 antigen and activity), and the euglobulin clot lysis time (ECLT). RESULTS: The only significant differences between the two groups occurred 6 h after surgery when the ECLT was longer (p < 0.005; Mann Whitney), and PAI-1 antigen and activity were higher (p < 0.01 and p < 0.001, respectively; Mann Whitney) after open cholecystectomy than laparoscopic cholecystectomy. CONCLUSIONS: Other changes in fibrinolysis and coagulation were similar for open and laparoscopic cholecystectomy. With respect to hemostasis, laparoscopic cholecystectomy does not increase the risk of thromboembolic complications compared to the conventional procedure.     

Activation of clotting factor XI without detectable contact activation in experimental human endotoxemia

Evidence of factor XI (FXI) activation in vivo is scarce. In addition, it remains uncertain whether thrombin, factor XIIa (FXIIa), or perhaps another protease is responsible for FXI conversion. We investigated the activation of FXI in eight healthy volunteers after infusion of a low dose of endotoxin (4 ng/kg of body weight). Activation of prekallikrein FXII, FXI, and prothrombin was measured with sensitive enzyme-linked immunosorbent assays (ELISAs), and FXI activation was measured with a novel enzyme capture assay that detects noncomplexed FXIa. Activation of FXI was apparent with a significant plasma peak level of noncomplexed FXIa of 10 to 11 pmol/L at 1 and 2 hours after endotoxin infusion, followed by a gradual increase in FXIa-FXIa inhibitor complexes, measured in the ELISAs, with a summit of 11 to 15 pmol/L at 6 and 24 hours, respectively. In accordance with previous studies, thrombin generation was detected 1 hour after endotoxin infusion to become maximal after 3 to 4 hours. In contrast, we did not find any evidence of contact activation, because markers of activation of prekallikrein and FXII remained undetectable. From the FXIa data a theoretical model was constructed which suggested that inhibition of FXIa does not take place in the plasma compartment, but is localized on a surface. These data provide the first evidence for FXI activation in low-grade endotoxemia and suggest that FXI is activated independently of FXII.     

Aprotinin inhibits plasmin-induced platelet activation during cardiopulmonary bypass

PURPOSE: We correlated the expression of bcl-2 with accumulation of p53 protein in bone marrow metastases from patients with androgen independent prostate cancer and a history of hormonal ablation therapy. These results were correlated with clinical parameters, including the extent of bone marrow metastases and patient survival. MATERIALS AND METHODS: All 43 patients studied had evidence of prostate cancer progression following androgen deprivation therapy and histologically confirmed bone marrow metastases. Decalcified tissue sections were used for immunohistochemical evaluation of bcl-2 protein and p53 protein accumulation. RESULTS: We previously established that p53 protein accumulation as detected by immunohistochemistry is a reliable indicator of p53 gene mutation in prostate cancer. Immunoreactivity was demonstrated for p53 protein in 22 of 43 cases and for bcl-2 protein in 14. A total of 28 cases (65%) exhibited immunohistochemical evidence of p53 and/or bcl-2 expression, and 15 (35%) were negative for p53 and bcl-2. The expression of bcl-2 and accumulation of p53 were independent events (p < 0.01). The expression of bcl-2 or accumulation of p53 protein in prostate cancer metastases did not significantly influence patient survival or the extent of metastatic disease. CONCLUSIONS: The presence or absence of p53 protein accumulation and/or bcl-2 expression did not correlate with tumor burden or patient survival in stage D androgen independent prostate cancer bone marrow metastases. The expression of bcl-2 protein occurs independently of and is inversely correlated with p53 mutations in advanced prostate cancer.     

Adenoviral vector-mediated interleukin-10 expression in vivo: intramuscular gene transfer inhibits cytokine responses in endotoxemia

Malacoplakia is a chronic inflammatory disease the etiology of which remains obscure. It has a very low incidence and affects primarily the genitourinary tract, although it has been described in some other organs. This paper presents a historic insight of the clinical cases diagnosed in this centre, and includes a review and update of several issues related to this entity such as pathogenesis, pathological anatomy and treatment. Also, the peculiarities related to the involvement of each separate organ with regard to diagnosis, prognosis and treatment are described.     

Effects of extensive oral surgery and hemorrhage on coagulation and fibrinolysis

The changes in coagulation and fibrinolytic activity in 22 patients with oral cancer undergoing extensive surgical procedures were studied. The patients were divided into two groups: group I patients suffered blood loss of less than 2,000 mL and group II patients had blood loss of more than 2,000 mL. The platelet count decreased significantly during surgery, at the end of surgery and on the 1st postoperative day in both groups. Fibrinogen was decreased during and at the end of surgery in both groups, but increased significantly on the 3rd postoperative day and reached about two times the preoperative levels on the 7th postoperative day. Fibrin degradation products increased significantly after surgery and reached the maximum value on the 1st postoperative day in both groups. Plasmin inhibitor complex and plasminogen increased significantly on the 3rd and 7th postoperative days. There was no clear evidence regarding the influence of blood loss on coagulation and fibrinolytic factors except for platelets. It was concluded that coagulation and fibrinolysis are enhanced between the 3rd and 7th postoperative days.