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Biomedical subjects

I A Reilly

Publications and source records attributed to I A Reilly.

17 recordsLinked to original sources
PubMed1992

Role of autocrine and paracrine production of granulocyte-macrophage colony-stimulating factor and interleukin-1 beta in the autonomous growth of acute myeloblastic leukaemia cells--studies using purified CD34-positive cells.

The blast cells from some patients with acute myeloblastic leukemia (AML) proliferate autonomously in vitro. We have previously identified four groups of AML blasts based upon their growth characteristics in vitro, in particular the degree of autonomous growth. We have now measured the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-1 beta (IL-1 beta) by AML cells with different growth characteristics, using two sensitive enzyme-linked immunosorbent assays. Our results show a correlation between the capacity of AML blasts to produce GM-CSF and IL-1 beta and the ability to grow autonomously in vitro. Blasts from cells with no autonomous growth (n = 5) secreted low or undetectable amounts of GM-CSF and IL-1 beta. Blasts with totally autonomous growth (n = 10) secreted the highest levels of GM-CSF (mean 2469 pg/10(3) cells) and IL-1 beta (mean 3156 pg/10(6) cells). Whereas blasts with partially autonomous growth (n = 9) secreted intermediate levels of GM-CSF (mean 270 pg/10(6) cells) and IL-1 beta (mean 931 pg/10(6) cells). In order to determine whether GM-CSF production was autocrine or the consequence of paracrine secretion by differentiated leukemic cells, we studied the degree of autonomous growth and production of GM-CSF by CD34-positive blasts from eight patients whose unfractionated cells produced GM-CSF. We found that CD34-positive blasts from six of these cases grew autonomously to a degree comparable to that of the unfractionated cells, and that CD34-positive blasts produced GM-CSF either autonomously or in response to recombinant IL-1 beta. Our data suggests that in the majority of cases, CD34-positive blasts are capable of autonomous growth and autocrine GM-CSF production, however this is variably regulated by the paracrine production of IL-1 beta by CD34-negative cells.

Antigens, CD↗
PubMed1990

Interleukin-1 is one factor which regulates autocrine production of GM-CSF by the blast cells of acute myeloblastic leukaemia.

The role of interleukin-1 (IL-1) as a regulator of the autonomous growth of the blast cells of acute myeloid leukaemia (AML) has been studied on samples isolated from 15 patients. In nine out of 10 patients with evidence of partial autonomous blast cell growth. IL-1 further stimulated cell growth in both suspension culture and in a clongenic assay. IL-1 also stimulated cell growth in two out of three cases with no evidence of autonomous growth whereas no additional stimulation was observed in two cases with totally autonomous growth. In blast cells stimulated by IL-1, synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) was increased and the proliferative response to IL-1 was inhibited by an antibody to GM-CSF. In all samples with evidence of autonomous growth, blast cell conditioned medium (BCCM) contained IL-1 activity (range 0.7-74.2 units ml) and a polyclonal antibody to IL-1 markedly inhibited autonomous growth in four samples. BCCM from three of these four samples contained GM-CSF, the synthesis of which was suppressed when BCCM was prepared in the presence of anti-IL-1. Our data suggests that endogenous IL-1 is an important factor in the regulation of the production of GM-CSF and hence of autonomous growth of AML blasts, but that other mechanisms regulating GM-CSF production may exist.

Cell Division↗
PubMed1989

Heterogeneous mechanisms of autocrine growth of AML blasts.

The ability of blast cells to grow autonomously and to produce autostimulatory growth factors has been investigated in 25 consecutive patients with AML. An autostimulatory index (ASI) was calculated (no. of colonies without CSF divided by no. of colonies with CSF) and patients classified into four groups: Group 1 (n = 3): non-growers; Group 2 (n = 4): CSF-dependent (ASI less than 0.1); Group 3 (n = 11): partially autonomous (ASI 0.1-0.8); and Group 4 (n = 7): fully autonomous/CSF-unresponsive (ASI greater than 0.8). In Group 3 patients colony formation and DNA synthesis were significantly (P less than 0.01) augmented by CSFs but at high cell concentrations became CSF-independent. Blast cell-conditioned medium (BCCM) from these patients exhibited potent autostimulatory activity, increasing DNA synthesis by less than or equal to 5-fold, and also stimulated CSF-dependent homologous blasts by less than or equal to 20-fold. In 5/5 this activity was neutralized by anti-GM-CSF, which also inhibited autonomous proliferation of their blast cells. Group 4 blasts also secreted GM-CSF but their BCCM possessed no autostimulatory activity, and anti GM-CSF failed to inhibit their autonomous growth. No membrane-associated CSF activity was found, however purified cytosolic fractions stimulated proliferation of CSF-dependent homologous blasts, consistent with production and secretion of CSF which is present in active form in the cytosol but does not autostimulate via membrane receptors. These results suggest that autocrine mechanisms are important in regulating blast cell proliferation, but that the mechanisms are heterogeneous.

Bone Marrow↗
PubMed1989

The role of cell contact and autostimulatory soluble factors in the proliferation of blast cells in acute myeloblastic leukemia.

The role of cellular interactions in stimulating leukemic cell proliferation was studied in AML. When peripheral blood blasts were cultured in suspension in flat-bottomed vessels at low cell numbers (15.6-500 x 10(3)/ml), the rate of DNA synthesis [( 3H]thymidine incorporation) was proportional to cell density. When cells were cultured under otherwise identical conditions in round-bottomed vessels, to induce cell crowding, the rate of DNA synthesis was independent of cell numbers and was significantly augmented (p less than or equal to 0.01). Physical separation of cells in round-bottomed (crowded) cultures by latex beads reduced DNA synthesis to the same rate as that in flat-bottomed (non-crowded) cultures. In contrast, addition of mitomycin-treated autologous blasts had little effect in crowded cultures but increased DNA synthesis in non-crowded cultures. DNA synthesis was also enhanced: by reducing culture volume to concentrate any diffusible factors produced by the cells or by blast cell conditioned medium from autologous blasts grown under crowded conditions. Cells cultured in suspension for 72 hr under crowded/non-crowded conditions both formed blast cell colonies in methyl cellulose; however, the number of colonies derived from crowded cultures was greater. These results indicate that cell proximity promotes proliferation of blasts and blast cell progenitors; both close cell contact and autostimulatory soluble factors are important in regulating growth of AML blasts.

Cell Adhesion↗
PubMed1988

Transferrin receptor expression of AML blasts is related to their proliferative potential.

Expression of the transferrin receptor (TfR) was studied on peripheral blood blast cells from 11 patients with acute myeloid leukaemia (AML). Using a monoclonal anti-TfR antibody (OKT9) and a polyclonal antibody against surface membrane-bound transferrin, a proportion of blasts from all the patients was found to express receptors for transferrin. Further analysis of OKT9 expression using a fluorescent activated cell sorter (FACS) showed that the TfR was heterogeneously distributed in the blast cell population. In five out of six samples studied, stimulation of DNA synthesis following short-term culture induced a several-fold increase in TfR display as analysed by flow cytometry using OKT9 or FITC-conjugated transferrin. Blasts from seven patients stained with OKT9 were separated on the FACS into positive and negative or weakly positive fractions. Culture of the TfR negative population in a blast cell colony assay produced no colonies in either of two patients. In a further five patients the colony forming cells were predominantly associated with the TfR strongly positive fraction (52 +/- 25 colonies/10(4) cells) rather than the TfR weakly positive fraction (12 +/- 11 colonies/10(4) cells). Analysis of colony size showed that clones derived from the weakly positive fraction were smaller than clones from the TfR strongly positive fraction. These results suggest that TfR display by AML blasts is related to their proliferative capacity and is expressed by the leukaemic stem cell fraction.

Antibodies, Monoclonal↗
PubMed1988

Leukotriene B4 synthesis and neutrophil chemotaxis in chronic granulocytic leukaemia.

A sensitive gas chromatography-mass spectrometric method was used to measure the generation in whole blood of leukotriene B4 (LTB4), a potent stimulator of neutrophil chemotaxis, in eight patients with chronic granulocytic leukaemia (CGL) and 12 healthy controls. LTB4 was detectable in unstimulated samples from all the patients (mean 194 (70 SEM) pg/ml), and the capacity for LTB4 production after stimulation with calcium ionophore (A23187) was similar in patients (32.1 (11) ng/10(6) leucocytes) and controls (38.1 (4) ng/10(6) leucocytes). In response to stimuli which induce neutrophil activation, LTB4 production was significantly greater in the patients than in controls: 35.6 (13) v 13.0 (3) ng/ml, p less than 0.05 (f-met-leu-phe); and 42.4 (16) v 14.7 (4) ng/ml, p less than 0.02 (opsonised zymosan). Anti-IgE stimulated considerably more LTB4 production in patients with CGL than in controls (3.86 (1.6) v 0.83 (0.43) ng/ml; p less than 0.005) and this correlated significantly (p less than 0.05) with the basophil count. Neutrophil chemotaxis to LTB4, however, was significantly impaired in the patients with CGL even at the highest concentration of LTB4 (10(-5) M). Chemotaxis to f-met-leu-phe, phagocytosis, and bacterial killing were normal. Thus although LTB4 synthesis is normal or even enhanced in patients with CGL, specific defects in LTB4-mediated responses may contribute to neutrophil dysfunction in this disease.

Adult↗
PubMed1988

Aspirin in cardiovascular disease.

Although other mechanisms may be contributory, the antithrombotic properties of aspirin derive predominantly from its platelet-inhibitory effects. These are mediated via irreversible acetylation of platelet cyclo-oxygenase with subsequent blockade of platelet thromboxane synthesis. Long term administration of doses of aspirin as low as 20mg daily depresses platelet thromboxane formation by more than 90%; however, higher doses appear to be necessary to prevent thromboxane-dependent platelet activation in vivo. While there is evidence of biochemical selectivity with low doses of aspirin, significant reduction of the platelet-inhibitory eicosenoid, prostacyclin, occurs even at dosages ranging from 20 to 40mg daily. The ability of aspirin to prevent the occurrence or recurrence of vaso-occlusion has been extensively investigated. In the secondary prevention of myocardial infarction 7 placebo-controlled trials involving more than 15,000 patients have been completed. The dose of aspirin varied from 300 to 1500mg daily. Although none of the individual trials produced statistically significant reductions in total or coronary mortality, taken together the results are highly suggestive of a beneficial effect of aspirin. Similarly, 2 recent studies in patients with unstable angina demonstrated a protective effect of aspirin against acute myocardial infarction and death. While each study employed widely different doses of aspirin (324mg and 1250mg daily) similar reductions in mortality were reported. The effects of aspirin on the prevention of coronary artery bypass graft occlusion have been evaluated in 9 trials. Aspirin in doses of 100 to 975mg daily was shown to be of benefit in preventing early (less than 6 months) graft occlusion, particularly when therapy was started within 24 hours of operation. In patients with prosthetic vascular grafts of the lower limbs, aspirin has been shown to reduce platelet deposition, however further controlled trials will be required to establish the patient population most likely to benefit and, as in all these studies, the optimum dose of aspirin to employ. In patients with prosthetic heart valves it is clear that aspirin alone is insufficient to prevent thromboembolic complications and when administered as an adjunct to anticoagulant therapy it is associated with a high incidence of bleeding. In contrast, there is convincing evidence from several studies for the efficacy of aspirin in doses of 990 to 1300mg daily in the prevention of stroke and death in patients with transient ischaemic attacks.(ABSTRACT TRUNCATED AT 400 WORDS)

Aspirin↗
PubMed1987

Inhibition of thromboxane formation in vivo and ex vivo: implications for therapy with platelet inhibitory drugs.

The capacity of platelets to generate thromboxane A2, reflected by measurement of serum thromboxane B2 (TxB2), greatly exceeds the systemic production of thromboxane in vivo. Thus, it is possible that substantial but incomplete inhibition of thromboxane formation ex vivo would still allow marked augmentation of thromboxane production in vivo. To address this hypothesis, we administered aspirin 120 mg, a selective inhibitor of thromboxane synthase (TxSl), 3-(1H-imidazol-1-yl-methyl)-2-methyl-1H-indole-1-propanoic acid (UK-38, 485) 200 mg, and a combination of both drugs to 12 healthy volunteers and measured the effects on serum TxB2 and urinary 2,3-dinor-thromboxane B2 (Tx-M), an index of endogenous thromboxane biosynthesis. Although serum TxB2 was maximally inhibited by 94 +/- 1% after aspirin and 96 +/- 2% after the TxSl, maximal depression of Tx-M was only 28 +/- 8% and 37 +/- 9%, respectively. Combination of aspirin with the TxSl resulted in a small but significant increase in inhibition of thromboxane generation ex vivo (98 +/- 1% v 94 +/- 1%; P less than 0.05), but a disproportionately greater fall in thromboxane synthesis in vivo (58 +/- 7%; P less than 0.01). Consistent with further inhibition of platelet thromboxane synthesis, addition of the TxSl abolished the transient decline in prostacyclin formation after aspirin alone. Administration of a lower dose of aspirin (20 mg) to 6 healthy subjects caused a small reduction in Tx-M (12 +/- 4%; P less than 0.05) and inhibited serum TxB2 by 48 +/- 2%. The relationship between inhibition of platelet capacity to form thromboxane ex vivo (serum TxB2) and synthesis in vivo (Tx-M) departed markedly from the line of identity. When total blockade of the capacity of platelets to generate thromboxane is approached, minor decrements in capacity result in a disproportionate depression of actual thromboxane biosynthesis. These results imply that pharmacologic inhibition of serum TxB2 must be virtually complete before thromboxane-dependent platelet activation is influenced in vivo.

Adult↗
PubMed1986

Biosynthesis of thromboxane in patients with systemic sclerosis and Raynaud's phenomenon.

Thromboxane A2, the predominant cyclo-oxygenase product of arachidonic acid in platelets, is a potent vasoconstrictor and platelet agonist. Analysis of urinary metabolites by gas chromatography and mass spectrometry is a specific non-invasive method of measuring the biosynthesis of thromboxane that avoids the problem of platelet activation ex vivo. Excretion of the major urinary thromboxane metabolite, 2,3-dinor-thromboxane B2, was significantly increased (p less than 0.001) in 10 patients (nine women) with systemic sclerosis complicated by Raynaud's phenomenon compared with healthy controls (486 (SD 88) v 162 (38) ng/g creatinine) and increased further in the patients (to 1007 (212) ng/g creatinine) during application of a cold stimulus sufficient to induce digital vasoconstriction. Consistent with an increase in platelet-vascular interactions in vivo, excretion of a prostacyclin metabolite was also significantly increased (p less than 0.005) in the patients with systemic sclerosis (248 (39) v 112 (10) ng/g creatinine) and tended to increase further on cooling. Biosynthesis of thromboxane is increased in patients with systemic sclerosis and may exacerbate digital vasospasm that such patients develop when cold. This observation and the concomitant increase in the formation of prostacyclin provide a rationale for evaluating compounds that prevent the synthesis of thromboxane A2 or inhibit its action while preserving the potential homoeostatic role of prostacyclin.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

In vivo indexes of platelet and vascular function during fish-oil administration in patients with atherosclerosis.

Populations that consume a diet rich in marine lipids may have a lower risk of atherosclerotic disease. Fish oil contains the N-3 polyunsaturated fatty acid eicosapentaenoate, and the biosynthesis of thromboxanes and prostacyclins from eicosapentaenoate (thromboxane A3 and prostaglandin I3), rather than from the usual precursor arachidonate (thromboxane A2 and prostaglandin I2), may help to reduce the risk. To examine this hypothesis, we studied the effect of eicosapentaenoate supplementation (10 g per day) for one month on the synthesis of thromboxanes and prostacyclins, as assessed by urinary metabolite excretion, in six patients with peripheral vascular disease and seven normal controls. Supplementation markedly increased the eicosapentaenoate content of phospholipids from red cells and platelets. Synthesis of the platelet agonist thromboxane A2, which was elevated in the patients at base line, declined by 58 percent during supplementation but did not reach normal values. The decline in thromboxane A2, which is synthesized from arachidonate, coincided with the formation of the inactive thromboxane A3, which is synthesized from eicosapentaenoate. A lower dose of eicosapentaenoate (1 g per day) was not sufficient to maintain the changes in thromboxane A2 synthesis. Platelet function was only moderately inhibited during eicosapentaenoate supplementation, consistent with incomplete suppression of thromboxane A2 synthesis. These studies show that a high dose of eicosapentaenoate alters the pattern of synthesis of thromboxanes and prostacyclins. However, effects comparable to those of aspirin require long-term administration in high doses. Whether other properties of fish oil might render it a more attractive antithrombotic therapy remains to be determined.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

Eicosenoid biosynthesis and platelet function with advancing age.

The pathogenesis of atherosclerosis, a major cause of age-related mortality, remains poorly understood. Although platelets and their products, including thromboxane A2, may be of importance in this process, little is known about eicosenoid biosynthesis and platelet function with increasing age. In order to address the hypothesis that platelet activation increases with age, we measured various indices of platelet function in a group of apparently healthy individuals over the age of 50 years. The circulating platelet aggregate ratio, plasma beta-thromboglobulin and threshold aggregating concentration of arachidonic acid were similar to those in healthy subjects aged less than 40 years. Although the bleeding time (168 +/- 24 vs 300 +/- 24 seconds) was significantly (p less than 0.001) shorter in the older volunteers this may be unrelated to platelet function and merely reflect age related changes in skin and/or vascular function. To further assess platelet and vascular function in vivo, we measured excretion of the major thromboxane and prostacyclin metabolites in urine, 2,3-donor-thromboxane B2 (Tx-M) and 2,3-dinor-6-keto-PGF1 alpha (PGI-M). Both Tx-M (223 +/- 22 vs 152 +/- 19 pg/mg creatinine; p less than 0.005) and PGI-M (198 +/- 21 vs 121 +/- 13 pg/mg creatinine; p less than 0.005) excretion were significantly higher in the older volunteers. These subtle but significant changes in eicosenoid biosynthesis are consistent with the presence of platelet activation in vivo increasing with age in apparently healthy individuals.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

Increased thromboxane biosynthesis in a human preparation of platelet activation: biochemical and functional consequences of selective inhibition of thromboxane synthase.

Although thromboxane A2 is a potent platelet agonist and vasoconstrictor in vitro, our knowledge of its pathophysiologic importance in human disease is limited. To facilitate the elucidation of its role in vivo, we sought to define a human syndrome in which pharmacologic interventions designed to inhibit the biosynthesis or biologic actions of thromboxane A2 might be appropriately assessed. Patients with severe peripheral vascular disease were selected on the basis of elevated plasma beta-thromboglobulin and circulating platelet aggregates and compared with healthy, age-matched control subjects. In addition to the platelet indexes, their bleeding time was shorter and excretion of 2,3-dinor-thromboxane B2, a noninvasive index of thromboxane formation in vivo, and 2,3-dinor-6-keto-prostaglandin F 1 alpha, the major urinary metabolite of prostacyclin, was markedly increased. A selective inhibitor of thromboxane synthase, imidazo (1,5-2) pyridine-5-hexanoic acid, was administered to these patients under randomized, double-blind, controlled conditions. Platelet aggregation ex vivo, the circulating platelet aggregate ratio, and the bleeding time were all unaltered, despite almost maximal inhibition of platelet thromboxane formation 1 hr after dosing. By contrast, pronounced inhibition of aggregation was observed when platelet cyclooxygenase was inhibited by aspirin. During long-term dosing with the synthetic inhibitor, inhibition of thromboxane biosynthesis was incomplete, which would permit continued thromboxane-dependent platelet aggregation to occur. However, the failure of enzyme blockade to influence platelet function at the time of maximal drug action, despite efficient inhibition of serum thromboxane B2, suggests that accumulation of proaggregatory endoperoxides is also likely to have contributed to the persistence of platelet activation. We have characterized a human preparation in which platelet activation coexists with increased thromboxane biosynthesis. In this setting, platelet activation persists despite long-term administration of a thromboxane synthase inhibitor in a dosing regimen representative of that employed in clinical trials. Prolongation of drug action and combination with antagonists of the shared endoperoxide/thromboxane A2 receptor may be necessary to assess the potential of selective inhibition of thromboxane synthase as a therapeutic strategy in man.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

The biochemical pharmacology of thromboxane synthase inhibition in man.

Selective inhibitors of thromboxane synthase have two theoretical advantages over inhibitors of the cyclooxygenase enzyme as potential antithrombotic compounds. First, they do not prevent formation of prostacyclin, a platelet-inhibitory, vasodilator compound, coincident with inhibiting thromboxane biosynthesis. Second, the prostaglandin endoperoxide substrate that accumulates in the platelet in the presence of thromboxane synthase inhibition may be donated to endothelial prostacyclin synthase at the site of platelet-vascular interactions (endoperoxide "steal"). Selective inhibition of thromboxane biosynthesis coincident with enhanced prostacyclin formation in vivo has been observed after administration of these compounds to man. Despite these attractive features and the efficacy of these compounds in diverse short-term animal preparations of thrombosis, investigations of their efficacy in human disease have proven disappointing. This may reflect on the importance of thromboxane A2 in the diseases that have been investigated. Alternatively, the lack of drug efficacy may have resulted from either incomplete suppression of thromboxane biosynthesis and/or substitution for the biological effects of thromboxane A2 by prostaglandin endoperoxides during long-term dosing studies. Given that selective inhibition of thromboxane formation can be approached with aspirin, the particular value of these compounds is dependent on enhancing prostacyclin formation. Aspirin inhibits thromboxane-dependent platelet activation, but many platelet agonists are likely to act in concert in vivo and prostacyclin inhibits platelet aggregation induced by both thromboxane-dependent and thromboxane-independent mechanisms. To test the hypothesis that thromboxane synthase inhibitors are efficacious in human disease, compounds of longer duration of action are required. Combination with antagonists of the prostaglandin/thromboxane A2 receptor may be necessary to reveal their full beneficial action.

Clinical Trials as Topic↗