Effect of interleukin-1 beta converting enzyme inhibitor on acute myelogenous leukemia progenitor proliferation

Interleukin-1 beta (IL-1 beta) converting enzyme (ICE) is a cysteine protease that specifically cleaves precursor IL-1 beta to its biologically active form. Recent studies have also implicated ICE in the induction of apoptosis in vertebrate cells. Because IL-1 plays a major role in acute myelogenous leukemia (AML) blast proliferation, we sought to investigate the effect of ICE inhibition on AML progenitors. To do this, we used bocaspartyl (benzyl) chloromethylketone (BACMK) an inhibitor designed to penetrate cells and bind covalently to the active site of ICE. Our preliminary experiments showed that incubation of activated peripheral blood cells with 2.5 mumol/L of BAMCK downregulated production of mature IL-1 beta but had no effect on tumor necrosis factor-alpha. To test the effects of the inhibitor on AML cells, we first used the OCI/AML3 cell line. We found that these cells produce IL-1 beta and bind the biotinylated cytokine and that IL-1 inhibitors, such as IL-1 neutralizing antibodies, IL-1 receptor antagonist, and soluble IL-1 receptors, specifically inhibit OCI/AML3 proliferation, indicating that IL-1 beta is an autocrine growth factor for OCI/AML3 cells. The ICE inhibitor suppressed OCI/AML3 growth in a dose-dependent manner (at 0.4 to 4 mumol/L) and downregulated mature IL- 1 beta production, as assessed by Western immunoblotting. Similar results were obtained with marrow aspirates from 16 AML patients. The ICE inhibitor suppressed proliferation of AML precursors (by up to 78%; mean, 44%) in a dose-dependent fashion at concentrations ranging from 0.4 to 5 mumol/L but not proliferation of normal marrow progenitors; the suppressive effect was reversed by IL-1 beta. Furthermore, incubation of AML cells with 4 mumol/L BAMCK downregulated the production of mature IL-1 beta, suggesting that the growth-inhibitory effect is mediated through suppression of the biologically active cytokine. Our data indicate that inhibition of ICE suppresses AML blast proliferation and suggest that ICE inhibitors may have a role in future therapies for AML.     

Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes: a means to enrich megakaryocytes based on membrane rather than physical characteristics

We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions. Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients. The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added. Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma. Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes. All maturation stages of morphologically recognizable megakaryocytes were aggregated. The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated). Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin. A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions. The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population. We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics. This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.     

Acute nonlymphocytic leukemia following etoposide and cisplatin combination chemotherapy for advanced non-small-cell carcinoma of the lung

Combination chemotherapy is frequently used in the therapy of advanced non-small-cell lung cancer (NSCLC), but late complications are rarely recognized because of the short survival of most patients. Of 119 patients with advanced NSCLC treated with cisplatin and other drugs, four patients developed acute nonlymphocytic leukemia (ANLL). All four patients received etoposide and cisplatin with or without vindesine. Leukemia was diagnosed at 13, 19, 28, and 35 months after start of treatment. Three patients had morphologic and/or cytogenetic features of acute leukemia with significant monoblastic involvement; the fourth patient had trilineage dysplasia and cytogenetic abnormalities more commonly associated with therapy-related leukemia. Detailed analysis of the subgroup who survived longer than 1 year (24 patients) suggests that high cumulative doses of etoposide are leukemogenic; the median etoposide dose was 6,795 mg/m2 (first year only) in the four leukemic patients compared with 3,025 mg/m2 in the 20 nonleukemic patients (P less than .01). The rate of ANLL was 0.30 per person-year after the first year (95% confidence limits 0.11 to 0.90), with a cumulative risk of 15% +/- 11% at 2 years, and 44% +/- 24% at 2.5 years. We conclude that high doses of etoposide are potentially leukemogenic, and can induce a syndrome with features of acute monoblastic leukemia de novo that is distinct from other secondary leukemias.     

Effect of modulators of erythropoiesis on the hemoglobinization of human erythroid cell cultures

A rapid spectrophotometric assay capable of detecting the hemoglobin content of 1000 mature erythrocytes has been utilized to quantitate the total hemoglobin synthesized by the progeny of circulating human erythroid progenitors in both the plasma clot and methylcellulose culture systems. The pronounced variation in the effect of different erythropoietin preparations on the hemoglobin content of cultured human peripheral blood bursts, previously described in a subjective manner, has been objectively quantitated. Further experiments demonstrated that both lymphocyte conditioned media and dexamethasone substantially increased the total hemoglobin synthesized by the progeny of cultured erythroid progenitors. The elevated amount of hemoglobin present in erythroid cultures containing either lymphocyte conditioned media and/or dexamethasone was due to both increased colony numbers and colony size. Assay of the total hemoglobin content per erythroid culture is an accurate, sensitive, measure of erythropoiesis in vitro and should be a valuable adjunct to the enumeration of BFU-E-derived erythroid colonies.     

Myocardial perfusion and angiographic findings in patients with paradoxical sinus deceleration during dobutamine technetium-99m sestamibi-gated SPECT imaging

AIMS: Dobutamine-gated SPECT imaging allows simultaneous perfusion and function assessment and is an alternative stress method for evaluating coronary artery disease in patients unable to perform treadmill exercise. In some patients, a paradoxical decrease in heart rate has been observed during high dose dobutamine infusion. The purpose of this study was to describe the paradoxical decrease in heart rate observed during dobutamine perfusion scintigraphy and determine its relation to inducible ischemia and angiographic coronary artery disease. METHODS AND RESULTS: We studied 52 patients who underwent dobutamine-gated (99m)Tc sestamibi SPECT imaging and coronary angiography within 30 days. Paradoxical deceleration was defined as a decrease in heart rate >/=5 beats/min lasting at least 3 min during dobutamine infusion. Perfusion was graded on a five-point scale (0 = normal; 4 = absent uptake) and wall motion on a four-point scale (0 = akinesia/dyskinesia; 3 = normal) using the 20-segment model. Significant coronary artery disease was defined as >/=50% narrowing of lumen diameter of a major epicardial artery. Paradoxical deceleration occurred in 10 patients (19%, 95% CI 8-30%). A decrease in systolic blood pressure [8 patients (80%), 95% CI 56-92%] with angina [5 patients (50%), 95% CI 23-71%] often accompanied the decrease in heart rate. All 10 patients with sinus deceleration had an inferior wall perfusion defect and 8 of them had a corresponding wall motion abnormality on gated images. Significant coronary artery disease was present in all 10 patients with sinus deceleration, with an increased incidence of right coronary artery stenosis (p = 0.007). CONCLUSIONS: Paradoxical deceleration observed during dobutamine perfusion scintigraphy is associated with inducible ischemia and angiographic coronary artery disease and the underlying mechanism for this phenomenon may be the activation of the Bezold-Jarisch reflex.     

Therapeutic angiogenesis with vascular endothelial growth factor in peripheral and coronary artery disease: a review

Therapeutic angiogenesis constitutes an alternative treatment for patients with extensive tissue ischaemia in whom primary vascular reconstruction procedures are not feasible or have previously failed. At present vascular endothelial growth factor (VEGF) has been the most widely used angiogenic factor in experimental and human clinical trials. Early clinical data provide evidence that gene transfer of the VEGF gene can achieve beneficial angiogenesis, with minimal side-effects. Ongoing phase III clinical studies will reveal definitive efficacy.     

The Association Between Hospital End-of-Life Care Quality and the Care Received Among Patients With Heart Failure

ContextImproving end-of-life care (EOLC) quality among heart failure patients is imperative. Data are limited as to the hospital processes of care that facilitate this goal.ObjectivesTo determine associations between hospital-level EOLC quality ratings and the EOLC delivered to heart failure patients.MethodsRetrospective analysis of the Veterans Health Administration (VA) and the Bereaved Family Survey data of heart failure patients from 2013 to 2015 who died in 107 VA hospitals. We calculated hospital-level observed-to-expected casemix-adjusted ratios of family reported excellent EOLC, dividing hospitals into quintiles. Using logistic regression, we examined associations between quintiles and palliative care consultation, receipt of chaplain and bereavement services, inpatient hospice, and intensive care unit death.ResultsOf 6256 patients, mean age was 77.4 (SD = 11.1), 98.3% were male, 75.7% were white, and 18.2% were black. Median hospital scores of “excellent” EOLC ranged from 41.3% (interquartile range 37.0%–44.8%) in the lowest quintile to 76.4% (interquartile range 72.9%–80.3%) in the highest quintile. Patients who died in hospitals in the highest quintile, relative to the lowest, were slightly although not significantly more likely to receive a palliative care consultation (adjusted proportions 57.6% vs. 51.2%; P = 0.32) but were more likely to receive chaplaincy (92.6% vs. 81.2%), bereavement (86.0% vs. 72.2%), and hospice (59.7% vs. 35.9%) and were less likely to die in the intensive care unit (15.9% vs. 31.0%; P < 0.05 for all).ConclusionPatients with heart failure who die in VA hospitals with higher overall EOLC quality receive more supportive EOLC. Research is needed that integrates care processes and develops scalable best practices in EOLC across health care systems.     

CD34+ progenitors and colony-forming units-granulocyte macrophage are recruited during large-volume leukapheresis and concentrated by counterflow centrifugal elutriation

The recruitment of mononuclear cells (MNCs), colony-forming units- granulocyte macrophage (CFU-GM), lymphocyte subpopulations, and CD34+ progenitor cells was studied during large-volume (15-25 L blood processed) peripheral blood stem cell (PBSC) harvests. Normal donors (n = 13) underwent a 4-hour leukapheresis designed to maximize PBSC yield (blood flow rate, 85 mL/min). Mean (+/− SD) volume processed was 17.7 +/− 0.4 L, and yield was 2.4 +/− 0.7 × 10(10) white cells containing 99 percent MNCs and 1.3 mL red cells per L of blood processed. Postapheresis hematocrit, platelets, and MNCs were reduced from preapheresis values by 7, 35, and 23 percent, respectively (p < 0.05). In nine donors, the component was collected as four 1-hour samples, and culturing of CFU-GM and flow cytometric analysis of lymphocyte subpopulations and CD34+/HLA-DR+ cells were done in individual samples. Total CFU-GM were 2.4 +/− 1.4 × 10(6) (3.0 +/− 1.8 × 10(4) CFU-GM/kg) and lymphocytes were 20.8 × 10(9), with 75 percent CD3+ T cells, 10 percent CD19/CD20+ B cells, and 17 percent natural killer cells. A more than twofold increase in CFU-GM and CD34+ cells was noted over the course of the 4-hour procedure (p < 0.05). In four donors, the leukapheresis component underwent counterflow centrifugal elutriation (CCE), which separated it into four fractions in an attempt to concentrate CD34+ and CFU-GM progenitors and to deplete T-lymphocytes on a large scale. There was a 1.8-, 4.6-, 3.9-, and 0.32-fold increase in CFU-GM in the four fractions relative to the unseparated component.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of plasma volume after storage of platelets in CP2D

BACKGROUND: There has been concern that further deterioration might occur if stored platelets are centrifuged to reduce their volume. Although such centrifugation appears to have minimal effect on platelets in CPDA-1 (osmolarity, 470 mOsm) there is no information on the situation with CP2D (580 mOsm). STUDY DESIGN AND METHODS: Platelet concentrates from CP2D packs were sampled at 1 and 5 days and after centrifugation was used to reduce the plasma volume to 10 mL. The aggregation, hypotonic shock response, morphology, pH, and lactate, glucose, pCO2 and pO2 levels were assessed, and values were compared to those seen with CPDA-1. In addition, blood was collected from the same donors into both CP2D and standard sodium citrate anticoagulant in an anticoagulant-to-blood ratio of 1:8 and the aggregation response of the fresh platelets was measured. RESULTS: Collection of blood into CP2D results in an immediate reduction of the platelet aggregation response when compared to that found after collection of blood into sodium citrate or CPDA-1. Aggregation is further decreased after storage; however, these changes and those for hypotonic shock, pH, lactate, glucose, and pCO2 are similar to those seen for CPDA-1. Additional centrifugation did not cause further change. CONCLUSION: Platelets stored in CP2D have reduced in vitro function after 5 days of storage, but subsequent centrifugation to reduce the plasma volume does not further alter these platelets.