Noninvasive evaluation of the bone marrow.
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Bone Marrow↗
Biomedical subjects
A Jakubowski
Publications and source records attributed to A Jakubowski.
Ischaemic cardiac hyperaemia: role of nitric oxide and other mediators.
In the perfused guinea-pig heart reactive hyperaemia (RH) after occlusion of coronary flow (1-60 s) was inhibited by 100-60% with NG-nitro-L-arginine (100 microM) and to a lesser extent (by 35%) after 8-phenyltheophylline (10 microM), but not by indomethacin (5 microM). Inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (5 microM) not only increased the concentration of adenosine in the coronary perfusate, but also prolonged the duration of RH. RH induced cardiac generation of prostacyclin, nitric oxide and adenosine as indicated by the appearance of 6-keto-PGF1 alpha, cyclic GMP, adenosine, inosine, hypoxanthine, xanthine and urate in the perfusate. Only NO and adenosine, but not prostacyclin, were responsible for RH. RH after short-term (1-10 s) coronary occlusion was mediated by NO, whereas adenosine and NO maintained RH that followed after longer (20 s-10 min) periods of cardiac ischaemia. Prostacyclin never participated in the mediation of RH.
6-Ketoprostaglandin F1 alpha↗
Vascular cell adhesion molecule-Ig fusion protein selectively targets activated alpha 4-integrin receptors in vivo. Inhibition of autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice.
The alpha 4 beta 1-integrin (CD49d, CD29) constitutively expressed on leukocytes regulates cell migration to inflammatory sites, cell activation, and development through its interactions with two alternate ligands, vascular cell adhesion molecule-1 (VCAM-1; CD106) expressed on cytokine-activated endothelium, dendritic and stromal cells, and the extracellular matrix protein fibronectin. Another alpha 4-integrin receptor, alpha 4 beta 7, expressed on leukocytes also binds VCAM-1 and fibronectin (FN), and controls homing to mucosal tissues through its interactions with mucosal vascular addressin MAdCAM-1. In vitro studies have shown that alpha 4-dependent cell adhesion is regulated by the activation state of the cell and by divalent cations. However, the existence and role of cells with different alpha 4 activation states in vivo have not been defined. Herein we show that a soluble ligand with the two N-terminal domains of human VCAM-1 fused to a human IgG1 constant region, VCAM-Ig, binds selectively to activated alpha 4-receptors on murine cells, such as those induced by Mn2+ in vitro. To determine whether the cells identified by VCAM-Ig were required under physiologic conditions, we assessed its anti-inflammatory effect. We show that VCAM-Ig is not bound to the majority of murine alpha 4+ cells after in vivo administration, yet it significantly delays the onset of adoptively transferred autoimmune diabetes. Thus, soluble VCAM-Ig can modify alpha 4-dependent disease progression, apparently by its selective action on cells with activated alpha 4-integrin receptors, thereby providing evidence for distinct alpha 4 activation states in vivo.
Animals↗
9-cis retinoic acid induces complete remission but does not reverse clinically acquired retinoid resistance in acute promyelocytic leukemia.
9-cis retinoic acid (RA) is a high-affinity ligand for both retinoic acid receptors (RARs) and retinoid "X" receptors (RXRs). Although all-trans RA does not bind to RXRs, RAR/RXR heterodimers or RXR/RXR homodimers bind to specific DNA response elements and modulate proliferation and differentiation of normal and malignant cells. Because the development of clinical resistance to all-trans RA has been associated with a progressive decrease in plasma drug concentrations, we evaluated the ability of 9-cis RA to induce in vitro cytodifferentiation in subclones of a retinoid-sensitive and resistant APL cell line (NB4) and in short-term cultures of fresh leukemic cells aspirated from patients. We also evaluated the clinical activity and pharmacokinetics of 9-cis RA (LGD 1057) in patients with APL who were previously treated with all-trans RA. In vitro tests of both retinoid-sensitive NB4 cells, as well as samples of fresh cells from 11 patients with APL, showed relatively equivalent degrees of sensitivity to both 9-cis RA and all-trans RA at concentrations ranging from 10(-6) to 10(-8) mol/L; however, no substantial cytodifferentiation was observed using either drug alone or in combination (10(-6) mol/L of each) in retinoid-resistant NB4 cells. Seven patients with APL who had previously relapsed from a remission induced by all-trans RA were treated with 9-cis RA at daily oral doses ranging from 30 to 230 mg/m2. Pharmacokinetic studies showed that the mean terminal plasma half-life of 9-cis RA (1.3 hours) changed very little after several weeks of dosing, although the mean change per dose level in area under the plasma concentration x time curves and peak plasma concentrations showed a decrease by 49% and 45%, respectively. Peak plasma concentrations equaled or exceeded concentrations that were effective against retinoid-sensitive cells in vitro. Despite these favorable pharmacokinetic results, only one of the seven patients achieved complete remission, corroborating in vitro studies of blasts from three of the nonresponders that showed a relatively equivalent degree of resistance to both retinoids. Our results suggest that while 9-cis RA may not induce its own catabolism to the same degree as all-trans RA, this feature does not appear to overcome clinically acquired resistance to all-trans RA in APL. Nonetheless, the drug can induce complete remissions in patients with APL and may be useful for extended therapy in other diseases. Future studies should address the use of lower doses in patients who have not previously received retinoid therapy.(ABSTRACT TRUNCATED AT 400 WORDS)
Administration, Oral↗
Treatment of chronic neutropenia associated with large granular lymphocytosis with cyclosporine A and filgrastim.
A patient with neutropenia and life-threatening infections secondary to T-gamma lymphoproliferative disease, who did not respond to treatment with recombinant human G-CSF (filgrastim), was treated with filgrastin plus cyclosporine A (CyA). The patient achieved a good response in the absolute neutrophil count and subsequently required a dose reduction in the filgrastim. The patient was eventually discontinued from the CyA but continues on filgrastim alone. While on therapy, the large granular lymphocytes disappeared from the circulation and the beta-TCR rearrangement, which was present prior to beginning therapy, became undetectable. The patient had no significant toxicity to the CyA or the filgrastim and he has not experienced any serious infections or required hospitalization. Filgrastim has proven to be relatively nontoxic and of some benefit to patients with this disease and should probably be utilized first when treatment is necessary. However, if improvement is not observed, these findings suggest that a trial of the combination of CyA plus filgrastim may be beneficial.
Bone Marrow↗
Vascular cell adhesion molecule (VCAM)-Ig fusion protein defines distinct affinity states of the very late antigen-4 (VLA-4) receptor.
The Very Late Antigen-4 receptor (VLA-4) (alpha 4 beta 1) is constitutively expressed on leukocytes and plays a role in cell trafficking, activation and development through its interaction with two alternative ligands, Vascular Cell Adhesion Molecule (VCAM-1) and fibronectin (FN). VLA-4-dependent cell adhesion is augmented by various stimuli, such as divalent cations, certain beta 1-specific monoclonal antibodies (mAbs) and cell activation. However, the steps of the adhesive process which they affect are currently undefined. In order to investigate whether or not these stimuli affect the primary step, VLA-4/ligand binding, we employed a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for VLA-4. Using this soluble ligand, we have directly demonstrated that the VLA-4 receptor can exist in at least three different affinity states on the cell surface. Two distinct high affinity states are induced on normal peripheral blood T cells, one by the anti-beta 1 mAb TS2/16, and one of 15-20 fold higher affinity by the divalent cation Mn2+. Interestingly, activation through the T cell receptor (TcR), through CD31 or by the Macrophage Inflammatory Protein-1 beta chemokine (MIP-1 beta) do not detectably increase VLA-4 affinity although they do augment VLA-4 dependent cell adhesion in vitro. Thus, VCAM-Ig binding defines high affinity VLA-4 receptors, revealing unique effects of the TS2/16 mAb and Mn2+ cations in vitro, and distinguishes VLA-4/VCAM interactions from subsequent steps in cell adhesion.
Animals↗
A pilot study of the biologic and therapeutic effects of granulocyte colony-stimulating factor (filgrastim) in patients with acute myelogenous leukemia.
In a carefully monitored pilot study, the in vivo biologic effects of filgrastim were investigated in eight patients with relapsed/refractory acute myelogenous leukemia. Within each patient, filgrastim was administered as a single agent prior to any chemotherapy in escalating doses of 0.12-6.0 micrograms/kg/day as a continuous intravenous infusion. The dose was increased every 14 days until an ANC of > or = 2500/mm3 had been achieved or there was evidence of proliferation of the leukemia. In patients who demonstrated growth of the leukemic clone, cytosine arabinoside was initiated at 200 mg/m2/day for 5 days. Through-out the course of therapy, the effects of filgrastim on maturation and proliferation were assessed by in vitro studies of bone marrow aspirates. Three patients demonstrated a sustained increase in ANC; one achieved a partial remission and remained on therapy for 31 weeks. Two of the three responding patients had hypocellular marrows at the time of initiating filgrastim and demonstrated a low but normal pattern of growth in CFU-GM assay early in the treatment course. This suggested that these two characteristics may define an environment in which filgrastim can induce a growth advantage for the normal residual hematopoietic elements. In this study of selected patients, filgrastim appeared safe.
Adult↗
Protection against adoptive transfer of autoimmune diabetes mediated through very late antigen-4 integrin.
The very late antigen-4 (VLA-4) integrin expressed on the surface of lymphocytes and macrophages can regulate their migration to inflammatory sites as well as control cellular activation. The role of VLA-4 in the establishment of autoimmune diabetes is not easily predicted given the multiplicity of adhesion pathways and their differential use by various cell types. The contribution of VLA-4 to insulin-dependent diabetes mellitus was investigated by administration of VLA-4-specific monoclonal antibodies (MoAb) in an adoptive transfer model of disease in NOD mice. This study shows that VLA-4-specific MoAbs profoundly inhibit the development of diabetes with protection sustained by repeated MoAb exposure. Insulitis was completely inhibited during treatment and progressed to a severe degree once MoAb treatment was suspended, yet approximately 40% of treated recipients failed to become diabetic during 1-2 months post-treatment. Although we cannot rule out depletion of a relatively minor subpopulation of cells by prolonged anti-VLA-4 MoAb exposure, this inhibition of diabetes onset by treatment with MoAbs to VLA-4 supports a dependence on VLA-4 for cellular functions leading to diabetes and demonstrates that a significant disease modifying effect can be mediated by targeting the VLA-4 integrin.
Animals↗
A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia.
Patients with idiopathic, cyclic, and congenital neutropenia have recurrent severe bacterial infections. One hundred twenty-three patients with recurrent infections and severe chronic neutropenia (absolute neutrophil count < 0.5 x 10(9)/L) due to these diseases were enrolled in this multicenter phase III trial. They were randomized to either immediately beginning recombinant human granulocyte colony-stimulating factor (filgrastim) (3.45 to 11.50 micrograms/kg/d, subcutaneously) or entering a 4-month observation period followed by filgrastim administration. Blood neutrophil counts, bone marrow (BM) cell histology, and incidence and duration of infection-related events were monitored. Of the 123 patients enrolled, 120 received filgrastim. On therapy, 108 patients had a median absolute neutrophil count of > or = 1.5 x 10(9)/L. Examination of BM aspirates showed increased proportions of maturing neutrophils. Infection-related events were significantly decreased (P < .05) with approximately 50% reduction in the incidence and duration of infection-related events and almost 70% reduction in duration of antibiotic use. Asymptomatic splenic enlargement occurred frequently; adverse events frequently reported were bone pain, headache, and rash, which were generally mild and easily manageable. These data indicate that treatment of patients with severe chronic neutropenia with filgrastim results in a stimulation of BM production and maturation of neutrophils, an increase in circulating neutrophils, and a reduction in infection-related events.
Adult↗
Interleukin-1 modulation of cytokine receptors on human neutrophils: in vitro and in vivo studies.
Interleukin-1 (IL-1) modulation of cytokine receptors (human IL-1 receptor [hIL-1R], human granulocyte colony-stimulating factor [hG-CSFR], human granulocyte-macrophage CSF receptor [hGM-CSFR], and human tumor necrosis factor receptor [hTNFR]) on human neutrophils was studied both in vitro and in vivo. In vitro, incubation of neutrophils with IL-1 at 37 degrees C for 0.5 or 8 hours caused a reduction of IL-1 binding in a dose-dependent manner, but did not demonstrably affect binding of the other cytokines tested. In vivo, neutrophils from patients with gastrointestinal malignancies who were participating in a clinical trial of recombinant human IL-1 beta (rhIL-1 beta) demonstrated modulation of cytokine receptors in an IL-1 beta dose- and time-dependent manner. At the two highest dose levels of IL-1 beta (0.068 and 0.1 microgram/kg), reduction (> 40%) of G-CSF binding and elevation (twofold to sixfold) of IL-1 binding to neutrophils was observed after 1 hour and 4 to 8 hours, respectively. In addition, IL-1 beta rapidly elevated G-CSF and glucocorticoid levels in plasma. Patients at the lowest dose level (0.002 microgram/kg) had a less dramatic change in these parameters. Further in vitro studies showed that synthetic glucocorticoids and G-CSF synergistically up-modulated IL-1 binding to neutrophils in a dose- and time-dependent manner. Scatchard analysis of binding data showed that this in vitro synergistic modulation was due to an increase in receptor numbers, rather than an increase in binding affinity. In addition, both human umbilical cord blood and bone marrow neutrophils responded to G-CSF and dexamethasone (Dex) with a superadditive increase in IL-1 binding. Therefore, one of mechanisms for IL-1 up-modulation of IL-1R on human neutrophils in vivo was due to the fact that IL-1 rapidly elevates serum levels of G-CSF and glucocorticoids.
Animals↗
Resolution enhanced NMR spectroscopy in biological systems via magnetic susceptibility matched sample immersion chambers.
A technique is described which reduced the magnetic susceptibility induced line broadening in NMR spectra obtained from three biological systems at 4.7 Tesla. Proton spectra from a sealed suspension of HL60 leukemic myeloblasts yielded minimum linewidths of 1.3 Hz at 200 MHz (0.0065 ppm) after 10 min of automated shimming. 31P spectra from an in vivo murine MCa mammary carcinoma yielded a well-resolved phosphorylcholine resonance without proton decoupling and with the resistive shim coil currents set to zero. 31P spectra from a perfused suspension of RIF-1 fibrosarcoma cells exhibited a gamma-nucleoside triphosphate resonance which was resolved into purine and pyrimidine components.
Animals↗
Interleukin-1: biological effects in human hematopoiesis.
Interleukin-1 (IL-1) produces many direct and indirect effects in hematopoiesis. Il-1 stimulates hematopoietic progenitors, and enhances production of other cytokines. Experimental data suggest a role in ameliorating the myelosuppression that occurs following cytotoxic chemotherapy. Recombinant human interleukin-1 has now entered clinical trial. We conducted a phase I trial of IL-1 beta alone, and following a myelosuppressive dose of 5-fluorouracil in patients with cancer. A prominent neutrophilia was noted within several hours, with a later, more sustained thrombocytosis. Hypotension was dose-limiting. We observed fewer days of neutropenia following 5-FU plus IL-1 compared to IL-1 alone, but the difference did not reach statistical significance. Early results from other investigators suggest a possible protective effect from chemotherapy-induced myelosuppression. Preclinical data suggest that combinations of IL-1 and lineage specific growth factors may produce synergistic hematologic effects.
Animals↗
Structure/function studies on vascular cell adhesion molecule-1.
Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. The major form of human VCAM1 contains seven extracellular Ig-like domains, with domain 1 designated as the most N-terminal. We have examined the relationship between human VCAM1 structure and function using a combination of domain truncation mutants and proteolytic fragmentation of recombinant soluble VCAM1. We have characterized two regions of VCAM1, localized to domains 4 and 5, which are highly sensitive to proteolytic cleavage, localized the epitope of the blocking monoclonal antibody 4B9 to domain 1, and found that domains 1-3 are sufficient for both its adhesive function and its ability to initiate T cell activation.
Amino Acid Sequence↗
Continuous treatment with all-trans retinoic acid causes a progressive reduction in plasma drug concentrations: implications for relapse and retinoid "resistance" in patients with acute promyelocytic leukemia.
Although all-trans retinoic acid (RA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), all groups have described clinical relapses despite continued RA treatment. This finding suggests that resistance to the cytodifferentiating effects of the retinoid had been acquired. To investigate potential mechanisms of clinical resistance to RA, we serially evaluated the clinical pharmacology of the drug in APL patients treated with this agent. Leukemic cells from patients relapsing from RA treatment were cultured in the presence of RA and examined for evidence of morphologic maturation. We also studied messenger RNA expression of the newly described gene product of the (15;17) translocation in APL, PML/RA receptor-alpha (PML/RAR-alpha). Serial pharmacokinetic studies showed that continuous daily RA treatment was associated with a marked decrease in plasma drug concentrations at the time of relapse compared with the initial day of therapy. Doubling the RA dose in six patients failed to reinduce response at the time of relapse and also failed to significantly augment plasma RA concentrations. However, leukemic cells obtained at the time of relapse from four patients retained in vitro sensitivity to the differentiating activity of RA (10(-6) mol/L). No change was observed in the pattern of PML/RAR-alpha expression assessed by Northern blot analysis at the time of relapse compared with pretreatment in two patients who were tested. These results indicate that clinical relapse and "resistance" to continuous treatment with all-trans RA in APL is associated with progressive reduction of plasma concentrations, potentially to levels below those that sustain differentiation of leukemic cells in vivo. Long-term success of this treatment will require the development of strategies that circumvent this pharmacologic phenomenon.
Carrier Proteins↗
A phase I trial of recombinant human interleukin-1 beta alone and in combination with myelosuppressive doses of 5-fluorouracil in patients with gastrointestinal cancer.
We studied escalating doses of recombinant human interleukin-1 beta (IL-1 beta) alone and after a myelosuppressive dose of 5-fluorouracil (5-FU) in patients with gastrointestinal cancer. Transient neutropenia, monocytopenia, and lymphocytopenia were observed followed by a 1.3- to 6.0-fold (mean, 3.46-fold) dose-dependent neutrophil leukocytosis (P less than .00001) on the days of IL-1 beta administration. Increases in platelet counts were observed at a median of 14 days (range, 6 to 23) after IL-1 beta administration. Transient hypoglycemia, rebound hyperglycemia, elevations in serum cortisol, and C-reactive protein were observed. Side effects included fever, rigors, and headache in the majority of patients. Hypotension was observed in three of five patients at the highest dose level (0.1 micrograms/kg) and was dose-limiting. Fewer days of neutropenia were noted after 5-FU plus IL-1 beta than after 5-FU alone; however, this difference did not reach statistical significance. These data show that IL-1 beta has stimulatory effects in human hematopoiesis.
Adult↗
Clinical pharmacology of deoxyspergualin in patients with advanced cancer.
Pharmacokinetic studies were carried out in 25 patients with advanced cancer receiving deoxyspergualin (DSG), a candidate anticancer agent, in a dose-finding Phase I study. The dosage range explored was 80 to 2160 mg/m2/day for 5 days by continuous i.v. infusion. The drug levels in plasma and urine were measured by high-performance liquid chromatography with postcolumn derivatization and fluorescence detection. One drug metabolite was demonstrated in plasma and urine of treated patients. This metabolite was extracted from urine and purified to homogeneity; thereafter, it was examined by high-performance liquid chromatography, nuclear magnetic resonance, and fragmentation mass spectrometry and was demonstrated to be identical to chemically synthesized desaminopropyl-DSG. The mean steady state plasma concentrations of DSG ranged from 0.28 to 11.1 microM at, respectively, the 80- and 2160-mg/m2 dosage levels. The plasma concentration at steady state and the area under the plasma concentration versus time curve of DSG were proportional to dose (r = 0.97). Following discontinuance of the infusion, DSG was cleared from the plasma in a biexponential fashion. The mean total body clearance was 364 +/- 78 ml/min/m2. Desaminopropyl-DSG was formed extensively at all dosage levels; mean steady state plasma levels of this metabolite reached a plateau 2.65 microM at a dose of 720 mg/m2/day and did not rise with further dose increments. The urinary content of DSG was examined in 20 patients over the dosage range from 160 to 960 mg/m2/day; in this group less than 10% of the administered dose was excreted as DSG. In four patients at the 720- and 960-mg/m2/day dosage levels, the total DSG plus metabolite excretion ranged from 7 to 18% of the administered dose, with comparable quantities occurring as the parent drug and desaminopropyl-DSG.
Animals↗
Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid).
BACKGROUND AND METHODS: Patients with acute promyelocytic leukemia have a characteristic (15;17) translocation, with a breakpoint on chromosome 17 in the region of the retinoic acid receptor-alpha (RAR-alpha). Since this receptor has been shown to be involved with growth and differentiation of myeloid cells in vitro, and since recent clinical studies have reported that tretinoin (all-trans-retinoic acid) induces complete remission in patients with acute promyelocytic leukemia we studied the effects of tretinoin on cellular maturation and molecular abnormalities in patients undergoing the induction of remission with this agent. RESULTS: Eleven patients with acute promyelocytic leukemia were treated with tretinoin administered orally at a dose of 45 mg per square meter of body-surface area per day. Nine of the 11 patients entered complete remission. In two patients, complete remission was preceded by striking leukocytosis that then resolved despite continued drug treatment. Serial studies of cellular morphologic features, cell-surface immunophenotypic analysis, and fluorescence in situ hybridization with a chromosome 17 probe revealed that clinical response was associated with maturation of the leukemic clone. All patients who responded to treatment who were tested by Northern blot analysis had expression of aberrant RAR-alpha. As patients entered complete remission, the expression of the abnormal RAR-alpha message decreased markedly; however, it was still detectable in several patients after complete morphologic and cytogenetic remission had been achieved. CONCLUSIONS: Tretinoin is a safe and highly effective agent for inducing complete remission in patients with acute promyelocytic leukemia. Clinical response to this agent is associated with leukemic-cell differentiation and is linked to the expression of an aberrant RAR-alpha nuclear receptor. Molecular detection of the aberrant receptor may serve as a useful marker for residual leukemia in patients with this disease.
Administration, Oral↗
Signaling by vascular cell adhesion molecule-1 (VCAM-1) through VLA-4 promotes CD3-dependent T cell proliferation.
Vascular cell adhesion molecule, VCAM-1, is an adhesion molecule expressed on activated endothelium thought to play a role in leukocyte migration to sites of inflammation. VCAM-1 adheres to leukocytes through the VLA-4 integrin. Recombinant soluble VCAM-1 (rsVCAM) and anti-CD3 mAb OKT3 were utilized to address the role of the VCAM-1/VLA-4 pathway in antigen-dependent T cell activation. Monocyte-depleted T cells proliferated upon exposure to co-immobilized OKT3 and rsVCAM but to neither alone. In contrast, an anti-VLA-4 mAb HP1/2 failed to co-activate with OKT3, despite the fact that both rsVCAM and HP1/2 support T cell adhesion comparably. These data indicate that adhesive function is not sufficient for co-stimulatory activity. They also reveal that VCAM-1 may play a role in regulating T cell immune responses as well as migration in vivo.
Antigens, CD↗