Platelet activation by fMLP-stimulated polymorphonuclear leukocytes: the activity of cathepsin G is not prevented by antiproteinases

Human polymorphonuclear leukocytes (PMN) activated by fMLP (in the presence of CaCl2, fibrinogen, and cytochalasin B) were able to induce aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production in coincubated autologousplatelets. Cell-free supernatants prepared from n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN were able also to induce platelet activation. Antibodies against cathepsin G and different serin protease inhibitors completely suppressed the activity of PMN-derived supernatants, indicating that cathepsin G is the major platelet activator released by PMN in our system. However, antiproteinases only partially affected platelet activation induced by PMN in mixed cell suspensions. Superoxide dismutase and catalase added to the cell suspension did not affect platelet activation nor potentiated serin protease inhibitors, making a role for short-lived oxygen radicals in our experimental system unlikely. Electron microscopic observation of stirred mixed cell suspensions preincubated for 2 minutes at 37 degrees C before stimulation showed a close PMN- platelets contact without any morphologic or biochemical event suggesting platelet activation. Preincubation of the cells without stirring to minimize PMN-platelet interaction before stimulation did not modify subsequent aggregation and platelet cytoplasmic Ca2+ increase in control samples. However, in this condition trypsin inhibitor from soybean completely prevented PMN-induced platelet activation. In samples preincubated without stirring in the presence of the antiproteinase, activated PMN stuck together but platelets preserved their discoid shape and did not appear significantly activated. We propose that membrane-to-membrane contact could create a microenvironment in which cathepsin G, discharged from stimulated PMN on adherent platelets, is protected from antiproteinases.     

Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen

Even if neoplastic cells express tumor associated antigens they still may fail to function as antigen presenting cells (APC) if they lack expression of one or more molecules critical for the induction of productive immunity. These cellular defects can be repaired by physiologic activation, transfection, or fusion of tumor cells with professional APC. Although such defects can be repaired, antitumor specific T cells may still fail to respond in vivo if they may have been tolerized. Here, human pre-B cell acute lymphoblastic leukemia (pre-B ALL) was used as a model to determine if primary human tumor cells can function as alloantigen presenting cells (alloAPC) or alternatively whether they induce anergy. In the present report, we show that pre-B cell ALL express alloantigen and adhesion molecules but uniformly lack B7-1 (CD80) and only a subset express B7-2 (CD86). Pre-B ALL cells are inefficient or ineffective alloAPC and those cases that lack expression of B7-1 and B7-2 also induce alloantigen specific T- cell unresponsiveness. Under these circumstances, T-cell unresponsiveness could be prevented by physiologic activation of tumor cells via CD40, cross-linking CD28, or signaling through the common gamma chain of the interleukin-2 receptor on T cells. Taken together, these results suggest that pre-B ALL may be incapable of inducing clinically significant T-cell-mediated antileukemia responses. This defect may be not only due to their inability to function as APC, but also due to their potential to induce tolerance. Attempts to induce clinically significant antitumor immune responses may then require not only mechanisms to repair the antigen presenting capacity of the tumor cells, but also reversal of tolerance.     

Hematologic and immunomodulatory effects of an interleukin-1 receptor antagonist coinfusion during low-dose endotoxemia in healthy humans

Endotoxin is a component of gram-negative bacteria that causes hematologic and immunologic changes through its induction of cytokines. Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of IL-1 that competes with IL-1 for occupancy of cell-surface receptors but possesses no agonist activity. We investigated the ability of human recombinant IL-1Ra to block the effects of low-dose endotoxin. Fourteen healthy male volunteers between 18 and 30 years old were injected intravenously with 3 ng/kg Escherichia coli endotoxin. Concurrent with the injections, nine volunteers received a 3-hour continuous intravenous infusion of IL-1Ra. The other five subjects were given a 3-hour infusion of saline. Volunteers injected with endotoxin experienced a threefold increase in circulating neutrophils over baseline. This neutrophilia was significantly reduced by 48% in subjects administered endotoxin plus IL-1Ra (P = .0253). Ex vivo mitogen-induced peripheral blood mononuclear cell proliferation decreased by greater than 60% at 3 and 6 hours after endotoxin injection (P = .0053). This endotoxin-induced reduction in mitogen response was reversed in subjects coinjected with IL-1Ra (P = .0253). Endotoxin-induced symptoms, fever, and tachycardia were unaffected by IL-1Ra. IL-1 appears to be an important mediator in endotoxemia because some of its hematologic and immunomodulatory effects can be blocked by IL-1Ra.     

The fate of the open canalicular system in surface and suspension- activated platelets

We have examined the movement of fibrinogen-gold (fgn-Au) complexes in platelets activated in suspension and by surface contact. Fgn-Au probes did not react with resting cells but were bound to the external membrane of platelets in suspension 5 seconds after addition of 1 U/mL of thrombin. At intervals over a period of 5 to 20 minutes, fgn-Au probes moved from the cell surface to peripheral and then deep channels of the open canalicular system (OCS). When platelets were surface activated by exposure to carbon-stabilized, formvar-coated grids for 5 to 20 minutes and then exposed to fgn-Au complexes for 5 minutes, probes were also observed in the OCS. At 5 minutes, over 40% of the platelets had concentrated fgn-Au in their OCS. Results after 10 minutes revealed 25% with gold-filled channels, 16% after 15 minutes, and 5% after 20 minutes. The decrease in frequency of OCS staining correlated with the increasing frequency of spread platelets, suggesting that tension produced by spreading may cause collapse of the OCS or that the OCS may evaginate onto the platelet during spreading. To evaluate the latter hypothesis, platelets were initially exposed to grids for 5 minutes and then incubated with fgn-Au for intervals of 5 to 20 minutes. The frequency of platelets with fgn-Au concentrated in the OCS was greatest at 5 minutes (44%) and decreased at the same rate as the frequency of spread platelets increased. Only 14.7% of the cells contained fgn-Au in the OCS after 20 minutes. These were primarily dendritic in form, while fully spread platelets rarely contained an OCS filled with the probe. The study indicates that fgn-Au particles are cleared to channels of the OCS independent of the mechanism of platelet activation. Fgn-Au that has been concentrated in the OCS at early stages of surface activation can be externalized during platelet spreading but remain internalized in suspension-activated cells. The OCS represents a membrane reservoir that can be evaginated onto the platelet surface during interaction with surfaces.     

Torsade de pointes: A nested case–control study in an integrated healthcare delivery system

BackgroundTdP is a form of polymorphic ventricular tachycardia which develops in the setting of a prolonged QT interval. There are limited data describing risk factors, treatment, and outcomes of this potentially fatal arrhythmia.ObjectiveOur goals were as follows: (1) to validate cases presenting with Torsade de Pointes (TdP), (2) to identify modifiable risk factors, and (3) to describe the management strategies used for TdP and its prognosis in a real‐world healthcare setting.MethodsCase–control study (with 2:1 matching on age, sex, and race/ethnicity) nested within the Genetic Epidemiology Research on Aging (GERA) cohort. Follow‐up of the cohort for case ascertainment was between January 01, 2005 and December 31, 2018.ResultsA total of 56 cases of TdP were confirmed (incidence rate = 3.6 per 100,000 persons/years). The average (SD) age of the TdP cases was 74 (13) years, 55 percent were female, and 16 percent were non‐white. The independent predictors of TdP were potassium concentration <3.6 mEq/L (OR = 10.6), prior history of atrial fibrillation/flutter (OR = 6.2), QTc >480 ms (OR = 4.4) and prior history of coronary artery disease (OR = 2.6). Exposure to furosemide and amiodarone was significantly greater in cases than in controls. The most common treatment for TdP was IV magnesium (78.6%) and IV potassium repletion (73.2%). The in‐hospital and 1‐year mortality rates for TdP cases were 10.7% and 25.0% percent, respectively.ConclusionsThese findings may inform quantitative multivariate risk indices for the prediction of TdP and could guide practitioners on which patients may qualify for continuous ECG monitoring and/or electrolyte replacement therapy.     

Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.