GM-CSF Priming of U937 Monocytic Cells Accelerates, But Does Not Increase, Tissue Factor (TF) Expression or Procoagulant Activity (PCA) After Stimulation with Endotoxin (LPS).

The monocytic U937 cell line, though immature, has many properties in common with mature monocytes. We have studied the antigenic expression and activity of tissue factor (TF) in the cytosol and on the surface of U937 cells after exposure to GM-CSF and endotoxin (LPS). Following exposure to LPS, both TF phenotype and procoagulant activity (PCA) increased linearly, with peak expression and activity after 18 hours of stimulation. Total PCA (tPCA) increased as early as 6 hours, unlike surface PCA (sPCA) which peaked at 18 hours. A linear correlation was observed between surface TF and both sPCA and tPCA. Incubation of cells with rHuGM-CSF did not effect phenotypic markers of monocyte maturation and had no significant effect on TF expression or PCA. However, cells activated with LPS after rHuGM-CSF priming, demonstrated accelerated expression of TF and PCA, with TF expression peaking at 6 hours and PCA at 2 hours. No increase in the absolute levels of TF were seen after priming with GM-CSF. We conclude that GM-CSF accelerates, but does not increase the magnitude of, the procoagulant response of monocytic cells to endotoxin. We propose that the initial accelerated PCA induction by LPS after rHu-GM-CSF priming, was due to conformational changes in TF and was not due to de novo synthesis of TF protein.

Administration of rHuGM-CSF activates monocyte reactive oxygen species secretion and adhesion molecule expression in vivo in patients following high-dose chemotherapy.

Microbicidal and cytocidal products of the respiratory burst and integrin adhesion molecule expression have been studied in monocytes from patients who received rHuGM-CSF during regeneration after high-dose chemotherapy. In this study, administration of rHuGM-CSF after high-dose chemotherapy significantly augmented the secretion of inducible products of the monocyte respiratory burst. Monocyte activation persisted for several weeks after the cessation of GM-CSF therapy. Under in vitro conditions that mimicked gram-negative (LPS) and gram-positive (opsonized Staphylococcus aureus) sepsis, the monocyte responded to such stimulation by exhibiting an enhanced release of hydrogen peroxide at both regeneration and several weeks later (P < 0.001). Similarly, GM-CSF administration significantly augmented the phenotypic expression of the beta 2-integrin adhesion molecules and allowed the leucocyte-specific selectin, LAM-1, and the beta 2-integrins to respond normally to inflammatory stimulation by LPS. We further present evidence that GM-CSF therapy restored the otherwise refractory status of monocytes to inflammatory stimulation that existed in those patients given chemotherapy alone. The restoration of monocyte responsiveness by GM-CSF following high-dose chemotherapy could be a potentially valuable and hitherto not described action of rHuGM-CSF on monocyte function. We conclude that administration of GM-CSF may have the potential for restoring as well as augmenting the anti-microbial and anti-tumour function of the monocyte after high-dose chemotherapy.

Modulation of CD13 expression during retinoic acid-induced differentiation of HL60 cells.

CD13/aminopeptidase-N, an enzyme expressed by myeloid cells, may be important in the regulation and signalling pathways that control myeloid growth and differentiation. In this study we have used the myeloid leukaemic cell line HL60, and its ability to differentiate when induced by all-trans-retinoic acid (ATRA), to study the regulation of CD13 molecules, and its associated aminopeptidase-N enzyme activity during the myeloid differentiation pathway. In addition, the effect of the growth factor granulocyte-macrophage colony stimulating factor (GM-CSF) on CD13 expression, by undifferentiated and differentiated HL60 cells, has been investigated. Our results show that CD13 expression, and its enzyme activity, is downregulated during differentiation of HL60 induced by ATRA, but not when using GM-CSF.

The effect of hydroxyethyl starch and other plasma volume substitutes on endothelial cell activation; an in vitro study.

OBJECTIVE: To study the effect of medium molecular weight hydroxyethyl starches on endothelial cell and neutrophil activation in vitro. SETTING: Laboratory analysis. METHODS: The effects of albumin and hydroxyethyl starch on the neutrophil adhesion molecule (CD11bCD18), with and without lipopolysaccharide stimulation, were studied in whole blood. E-selectin expression on human umbilical vein endothelial cells was stimulated with lipopolysaccharide alone and in the presence of either albumin or hydroxyethyl starch. The effect of albumin and hydroxyethyl starches on rapid endothelial cell activation was studied using von Willebrand factor release as a marker. MEASUREMENTS AND RESULTS: Hydroxyethyl starches but not albumin inhibited stimulated vWF release in a dose dependent manner. No effect was seen on endothelial E-selectin or neutrophil CD11bCD18 expression. CONCLUSIONS: these results suggest a possible beneficial role of hydroxyethyl starches in the inhibition of endothelial activation thus preventing neutrophil adhesion during sepsis syndrome.

Structure-dependent antitumor activities of novel anthracyclines YM1, YM3, YM4 and YM6: drug transport properties and effects on biomacromolecule synthesis in drug sensitive and resistant leukemia cells.

The antitumor activities of four novel doxorubicin (DOX) analogues, YM1, YM3, YM4 and YM6 in relation to their structure and drug transport properties, have been investigated in U937 monocytic and CCRF-CEM lymphoid drug sensitive leukemia cell lines, as well as in CEM/VLB100, a drug resistant subline displaying high levels of P-glycoprotein. Treatment of all cell lines with YM1, 3, 4 and 6 produced a dose-dependent decrease in DNA, RNA and protein synthesis as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. YM1 was more effective than YM3, YM4 or YM6 against the drug sensitive cells. The antitumor effects of all these DOX-analogues on macromolecule synthesis in U937 and CCRF-CEM cells were lower than that of DOX and epirubicin (EDR). A rapid accumulation of the novel anthracyclines was found in all cell lines compared with DOX or EDR. However, the maximal accumulation of the DOX-analogues was lower than that of EDR. There is a greater efflux from CCRF-CEM sensitive cells and less from CEM/VLB100 resistant cells of the DOX-derivatives when compared with EDR and DOX. Drug-induced cytotoxicity significantly correlated (P < 0.05) with drug retention levels in CCRF-CEM and U937 drug sensitive cells as indicated by an inverse correlation curve between anthracycline retention and drug-induced IC50 value. It was demonstrated that an increased level of drug retained within the sensitive cells would therefore produce a more cytotoxic effect of the drug. However, no such correlation was observed in CEM/VLB100 resistant cells. YM3 was shown to have an increased antitumor activity against CEM/VLB100 resistant cells compared with DOX with a lower resistance factor. These results showed that the antitumor effects of four novel DOX-analogues, like DOX or EDR, were associated with inhibition of DNA replication, transcription and translation. The finding that resistant leukemic cells are more susceptible to the cytotoxic effect of YM3 than DOX warrants further investigation to identify the intrinsic mechanism of resistance.

von Willebrand factor as a marker of endothelial cell activation following BMT.

Endothelial cell activation may play a role in thrombotic complications of BMT such as hepatic veno-occlusive disease (VOD), right atrial line thrombosis and microangiopathic haemolysis. To assess this, von Willebrand factor antigen (vWF:ag) was measured in 72 patients (25 allografts, 46 autografts and one syngeneic) during the first 6 weeks post-transplant. There was a significant rise in vWF:ag in both allografts and autografts but a greater increase was seen in the allografts. The changes in vWF:ag did not correlate with changes in C reactive protein showing that this was not merely an acute phase response. vWF multimers were normal in a subgroup of uncomplicated transplants showing that there was no large scale endothelial cell disruption. Patients with VOD did not have changes in vWF:ag that were consistently different from uncomplicated controls. Three of four patients who developed line thrombosis had higher levels of vWF:ag compared with control groups; multimeric structure of the vWF was again normal. These results show that there is endothelial cell activation post-BMT and that this is greater in allografts compared with autografts, thus suggesting a possible mechanism for the higher incidence of VOD in this group. There were no useful predictive markers of VOD or thrombosis in individual patients.

Streptokinase induced defibrination assessed by thrombin time: effects on residual coronary stenosis and left ventricular ejection fraction.

OBJECTIVE: To evaluate laboratory markers of defibrination early after thrombolytic therapy and to determine their relation to residual stenosis and left ventricular ejection fraction measured angiographically before discharge from hospital. DESIGN: Prospective analysis of defibrination after streptokinase measured by fibrinogen assay and thrombin time to provide a comparison of these coagulation variables for predicting angiographic responses to treatment in patients with acute myocardial infarction. SETTING: The coronary care unit of a district general hospital. PATIENTS: 44 patients with acute myocardial infarction treated by streptokinase infusion, all of whom underwent paired blood sampling before and one hour after streptokinase and cardiac catheterisation at a median of six (interquartile range 3-9) days later. MAIN OUTCOME MEASURES: Assay of thrombin time and plasma fibrinogen concentrations one hour after streptokinase infusion. Relations between these coagulation variables and residual stenosis in the infarct related coronary artery and left ventricular ejection fraction. Separate analyses are presented for all patients (n = 44) and those with patency of the infarct related artery (n = 35). RESULTS: Streptokinase infusion produced profound defibrination in every patient as shown by changes in thrombin time and circulating fibrinogen. Thrombin time after streptokinase infusion correlated significantly with both residual stenosis (r = -0.43, p < 0.005) and left ventricular ejection fraction (r = 0.38, p < 0.02). The importance of these correlations was emphasised by the interquartile group comparison which showed that a thrombin time > or = 49 seconds predicted a residual stenosis of 74% and an ejection fraction of 65%, compared with 90% and 49% for a thrombin time < or = 31 seconds (p < 0.01). When the analysis was restricted to patients with patency of the infarct related artery, the correlation between thrombin time and residual stenosis remained significant and group comparisons continued to show that patients in the highest quartile range had more widely patent arteries and better preservation of ejection fraction. Analysis of the fibrinogen data, on the other hand, showed insignificant or only marginally significant correlations with these angiographic variables. CONCLUSIONS: Early after streptokinase infusion for acute myocardial infarction, the level of defibrination measured by thrombin time has an important influence on residual coronary stenosis and left ventricular ejection fraction at discharge from hospital, values above 49 seconds being associated with the best angiographic result.

Thrombolytic treatment in acute myocardial infarction: neutrophil activation, peripheral leucocyte responses, and myocardial injury.

OBJECTIVE: To examine early leucocyte responses and neutrophil activation in acute myocardial infarction treated by streptokinase and to relate the findings to coronary recanalisation and indices of myocardial damage in order to provide further information about the role of neutrophils in the evolution of injury. DESIGN: Group analysis of paired blood samples, obtained before streptokinase treatment and one hour after it, and of three indirect measures of myocardial injury: left ventricular ejection fraction, QRS score, and peak creatine kinase. SETTING: The coronary care unit of a district general hospital. PATIENTS: 39 patients with acute myocardial infarction who underwent paired blood sampling (before streptokinase and one hour after streptokinase) and cardiac catheterisation 5 (3-8) days later. END POINTS: Changes in peripheral white cell and neutrophil counts and plasma elastase one hour after streptokinase infusion. Comparison of these variables in patients with and without patency of the infarct related coronary artery. Correlations between these variables and indirect measures of myocardial injury. RESULTS: Neutrophil activation, as reflected by plasma elastase, increased sharply one hour after streptokinase. Total white cell and neutrophil counts also increased. Changes tended to be more pronounced in patients with patency of the infarct related artery, though the trend was not statistically significant. Neutrophil activation before streptokinase was unrelated to indirect indices of myocardial injury but only one hour after streptokinase a weak negative correlation with left ventricular ejection fraction had developed. Peripheral neutrophil responses showed a similar relation to ejection fraction and also correlated with peak creatine kinase and QRS score. CONCLUSIONS: Thrombolytic treatment in acute myocardial infarction is associated with an abrupt reactive neutrophil response which provides an early measure of injury. It is also associated with neutrophil activation, probably in response to coronary recanalisation and myocardial reperfusion. Activated neutrophils are recognised as mediators of reperfusion injury in experimental infarction and the data in the present study provide preliminary evidence of a similar pathogenic role in the clinical setting.

Absorption of low and therapeutic doses of ferric maltol, a novel ferric iron compound, in iron deficient subjects using a single dose iron absorption test.

Ferric maltol is a novel ferric iron compound with potential use as an oral therapy for iron deficiency anaemia. Using a single, low dose iron absorption test we compared absorption of ferric maltol with absorption of ferrous sulphate in 21 iron deficient subjects. Absorption of 10 mg of ferric maltol as either aqueous solution or a single tablet compared favourably with that of an equivalent dose of ferrous sulphate. At a higher, more therapeutic dose of 60 mg elemental iron as tablets, absorption of ferric maltol appeared to be both more rapid and total absorption greater, than that seen with ferrous sulphate. We conclude that iron from ferric maltol, both at low dose and higher, more therapeutic doses, is at least as well absorbed as from ferrous sulphate. Ferric maltol is the first ferric iron formulation to be absorbed to a degree equivalent to that of ferrous iron salts and may represent a viable form of administration for ferric iron in the treatment of iron deficiency anaemia.

Combination chemotherapy with conservative obstetric management in the treatment of pregnant patients with acute myeloblastic leukaemia.

We have reviewed the management of pregnant women presenting with acute myeloblastic leukaemia (AML) at the London Hospital since 1972. Six women in the second or third trimester were diagnosed with AML over this period. One woman had termination of pregnancy at presentation in the second trimester. Three of the remaining five patients achieved complete remission following chemotherapy during pregnancy. Delivery was achieved by the vaginal route in three and by caesarean section in one patient. All were livebirths but one infant had Down's syndrome. Median maternal survival was 16 months (range 0-44 months). Long-term survival was achieved for both mother and infant in only one case. Longer maternal survival was seen in patients treated in the period 1980-1985. Increased survival appears to be related to the introduction of more aggressive chemotherapy schedules and improved supportive care.

Detection of granulocyte antibodies using flow cytometric analysis of leucocyte immunofluorescence.

Antigranulocyte antibodies are involved in the pathophysiology of a number of clinical disorders, which include: febrile transfusion reactions, severe pulmonary reactions to transfusion, auto-immune neutropenia, drug-induced neutropenia, and iso-immune neonatal neutropenia. Owing to the inherent difficulties of manipulating granulocytes in vitro, many of the serological techniques described for the detection of antigranulocyte antibodies are complex and sometimes difficult to reproduce. We describe the detection of alloreactive granulocyte antibodies using flow cytometric analysis of donor leucocyte suspensions in an indirect immunofluorescent test. The technique provides a semiquantitative detection of granulocyte antibodies in two groups of patients studied and, by providing as a comparison the reactivity on the corresponding mononuclear leucocytes, allows the distinction between granulocyte-specific antibodies and antibodies directed against the histocompatibility antigens.

Safety of intravenous immunoglobulin for treatment of auto-immune thrombocytopenia.

The incidence of hepatitis and HIV seroconversion has been examined in 64 patients receiving intravenous immunoglobulin (pepsin-treated at pH 4.0) for auto-immune thrombocytopenia. No evidence of HIV seroconversion has been detected. Five patients developed abnormal liver function following treatment. However, in no case could this be directly attributed to the treatment and no patient has developed chronic liver disease.