Hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders: studies of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and Flt-3 ligand (Flt3L).

The levels of hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders were investigated using a variety of approaches. Firstly, serum levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and Flt3-ligand (Flt3L) were examined in acute leukemia patients with treatment-induced cytopenia and complicating bacterial infections. Increased serum levels of both G-CSF and Flt3-ligand (Flt3L) were detected when these patients developed therapy-induced leukopenia, whereas GM-CSF levels were low or undetectable. Development of complicating bacterial infections then increased the serum levels of both G- and GM-CSF, and the Flt3L levels remained high during the infections. Secondly, release of growth factors was characterized for clonogenic T cells that remained in the circulation of acute leukemia patients with chemotherapy-induced cytopenia. CD4(+) and CD8(+) T cells from these patients released high levels of GM-CSF, relatively low levels of IL-3 secretion having been detected, and only a minority of the clones released detectable amounts of Flt3L. Thus, circulating T cells may contribute to the high systemic growth factor levels in cytopenic patients. Thirdly, plasma levels of GM-CSF and interleukin-3 (IL-3) were examined in patients with malignant disorders who received chemotherapy plus G-CSF for stem cell mobilization. Increased levels of GM-CSF and Flt3L were detected both in the patients' plasma and in the stem cell grafts. Despite the increased growth factor levels in neutropenic patients with complicating infections, the occurrence of febrile neutropenia did not have a major impact on normal hematopoietic reconstitution (i.e. duration of treatment-induced neutropenia) after intensive chemotherapy for acute myelogenous leukemia.

Platelet-derived growth factor (PDGF) in human acute myelogenous leukemia: PDGF receptor expression, endogenous PDGF release and responsiveness to exogenous PDGF isoforms by in vitro cultured acute myelogenous leukemia blasts.

We investigated effects of Platelet-derived growth factor (PDGF) and Platelet factor 4 (PF-4) on the functional characteristics of native, human acute myelogenous leukemia (AML) blasts. AML blast expression of the PDGF-receptor alpha-chain was detected for a subset of patients (45%), whereas PDGF-receptor beta-chain expression was detected for most patients (90%). Constitutive AML blast release of the PDGF-AB isoform (the major form also derived from normal platelets) was detected for 43% of patients, whereas PDGF-BB release was not detected for any patient. The PDGF isoforms AA, AB and BB had dose-dependent and divergent effects on spontaneous and cytokine-dependent AML blast proliferation, whereas for constitutive cytokine secretion (IL-1beta, IL-6, TNF-alpha) inhibitory effects were rare and all three isoforms usually had no effect or enhanced the constitutive secretion. The PDGF effects were caused by a direct effect on the AML blasts and were not dependent on the presence of serum. The PDGF effects could also be detected after in vitro culture of AML cells in the presence of IL-4+ granulocyte-macrophage colony stimulating factor. PF-4 had divergent effects on proliferation and cytokine secretion by native AML blasts. Our results suggest that exogenous (e.g. platelet-secreted) PDGF and PF-4 can function as regulators of leukemic hematopoiesis and possibly also modulate the function of residual AML cells in peripheral blood stem cell grafts. On the other hand, endogenous release of PDGF-AB by native blasts may modulate the function of normal cells in the bone marrow microenvironment (e.g. bone marrow stromal cells).

Effects of normal platelets on proliferation and constitutive cytokine secretion by human acute myelogenous leukaemia blasts.

The effects of soluble E-selectin, P-selectin and normal platelets on acute myelogenous leukaemia (AM L) blasts were investigated in vitro. We investigated effects on spontaneous and cytokine-dependent blast proliferation, and constitutive blast secretion of different cytokines. The presence of normal platelets during in vitro culture caused a dose-dependent increase in both spontaneous and cytokine-dependent AML blast proliferation. Addition of platelets also increased constitutive blast secretion of Interleukin 1beta (IL1beta ), IL6, GM-CSF and TNFalpha, whereas platelets had no effect on the release of IL1 receptor antagonist. The effects of platelets on constitutive cytokine secretion were also detected when platelets and AML blasts were cultured in different chambers separated by a permeable membrane, and a further enhancement was achieved when blasts and platelets were cultured together. Soluble P-selectin had no effect on constitutive AML blast cytokine secretion or the platelet-induced enhancement of the secretion. However, both soluble E- and P-selectin altered AML blast proliferation for a minority of patients. We conclude that normal platelets can modulate the function of human AML blasts in vitro.

In vitro culture of acute myelogenous leukemia blasts: a comparison of four different culture media.

Proliferative responses and cytokine secretion were compared when AML blasts were cultured in the three serum-free media, X-Vivo 10, X-Vivo 15, and defined serum-free medium (IMDM with mercaptoethanol, low-density lipoprotein, albumin, and transferrin) and in media containing 10% inactivated fetal bovine serum (FBS). The following AML blast functions were investigated: (a) constitutive cytokine secretion, (b) autonomous and cytokine-dependent proliferation, and (c) accessory cell function during T cell activation. Constitutive cytokine secretion and accessory cell function differed markedly when using different culture media. For the constitutive AML blast secretion of IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, no qualitative differences were seen, but quantitative differences were observed with decreased cytokine levels for cells cultured in X-Vivo 10 and X-Vivo 15. The accessory cell function of AML blasts was also decreased in the X-Vivo media, whereas differences were less pronounced when comparing AML blast proliferation. Our results clearly demonstrate that a well-characterized culture system is essential for in vitro studies of AML blast functions.

Evaluation study of the booklet "Preschoolers and Poisons".

Accidental poisonings remain a serious problem among young children. The booklet, "Preschoolers and Poisons," is part of the effort to educate the parents and caretakers to create a safe environment for their children. A pre-experimental, pre- and post-test, one group design, was employed with the purpose of determining if mothers of young children seen in St Paul Ramsey Medical Center's Pediatric Clinic had a gain in knowledge about accidental childhood poisoning and its prevention, and if they initiated preventive behavior after reading "Preschoolers and Poisons." The mothers' evaluation of the booklet was also solicited. A personal interview was used to collect data from a convenience sample of 40 mothers. The pre-test took place in the clinic, and the post-test over the phone, with an interim of 2 to 3 weeks. All participants read the booklet; 60% read the whole booklet and 40% read sections of interest. The mothers had a significant increase in knowledge, and a relationship between gained knowledge and age of the youngest child was found. The data showed that the Caucasian mothers in the study had a higher level of knowledge about accidental childhood poisonings and its prevention both before and after reading the booklet. Almost half of the mothers initiated preventive behavior. There was a positive relation between the initiation of preventive behavior and the age of the mother. Evaluations of the booklet were positive. Only 10% noted negative aspects such as the booklet being too short and missing a list of poisonous plants, while 80% identified positive aspects, such as the use of bright colors and the organization by age.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral blood progenitor cell grafts contain high levels of platelet-secreted mediators.

BACKGROUND: The cytokine network in peripheral blood progenitor cell (PBPC) grafts may affect hematopoietic reconstitution or the risk of postransplant relapse of malignant disorders through effects on normal progenitor cells or contaminating malignant cells. Whether thrombopoietin (TPO), SCF, and platelet-secreted mediators are parts of this network was investigated. STUDY DESIGN AND METHODS: Peripheral blood and PBPC plasma samples were collected consecutively from patients with malignant disorders who underwent PBPC harvest. Blood samples were collected immediately before and after apheresis. Patients underwent mobilization by chemotherapy plus G-CSF, except for one patient who received only G-CSF. Plasma levels were also determined for healthy controls. RESULTS: PBPC grafts had greater levels of platelet-secreted platelet factor 4 (PF4), beta-thromboglobulin, and platelet-derived growth factor isoform AB, as compared with venous levels in patients and controls. Although platelet and PF4 levels in autografts were significantly correlated, the graft:blood ratio was higher for PF4 than for platelets. In both the patients' blood and the autografts, TPO levels were increased from the levels in normal controls. Blood and graft levels of SCF were within the normal range. CONCLUSION: The cytokine network of PBPC autografts includes increased levels of TPO and several platelet-derived mediators.

Effects of vascular endothelial growth factor on acute myelogenous leukemia blasts.

Vascular endothelial growth factor (VEGF) and its specific receptors are expressed by various malignant cells, including acute myelogenous leukemia (AML) blasts. In this study we performed a detailed characterization of VEGF effects on native human AML blasts derived from a large group of consecutive AML patients with high blast counts in peripheral blood. Exogenous VEGF had divergent effects on spontaneous proliferation and cytokine-dependent (GM-CSF, G-CSF, IL-3) proliferation. Increased, decreased, or unaltered proliferation was observed in the presence of VEGF for various patients, and the VEGF effect differed even in the same patient depending on which exogenous cytokine being present together with VEGF. Similarly, increased, decreased or unaltered interleukin-1beta (IL-1beta) and IL-6 secretion was detected when VEGF was added, and for certain patients the effect of VEGF differed between IL-1beta and IL-6. Exogenous VEGF could also modulate proliferation and differentiation of clonogenic AML progenitors. Constitutive AML blast secretion of VEGF was detected for 40% of patients. Leptin, Flt3-L, IL-4, IL-10, and IL-13 had divergent effects on VEGF release by AML blasts. These results suggest that VEGF can modulate AML blast functions in vivo for a subset of patients. Furthermore, the detection of VEGF in peripheral blood stem cell (PBSC) autografts suggests that VEGF may influence the proliferation and possibly also the survival of contaminating AML cells in PBSC autografts. We conclude that VEGF may influence the functional characteristics of AML cells. Our results suggest that VEGF is important in leukemic hematopoiesis, and the detection of VEGF in PBSC autografts indicates that VEGF may influence the functional phenotype of contaminating AML cells in these grafts.

Platelet-derived growth factor (PDGF) in human acute myelogenous leukemia: PDGF receptor expression, endogenous PDGF release and responsiveness to exogenous PDGF isoforms by in vitro cultured acute myelogenous leukemia blasts.

We investigated effects of Platelet-derived growth factor (PDGF) and Platelet factor 4 (PF-4) on the functional characteristics of native, human acute myelogenous leukemia (AML) blasts. AML blast expression of the PDGF-receptor alpha-chain was detected for a subset of patients (45%), whereas PDGF-receptor beta-chain expression was detected for most patients (90%). Constitutive AML blast release of the PDGF-AB isoform (the major form also derived from normal platelets) was detected for 43% of patients, whereas PDGF-BB release was not detected for any patient. The PDGF isoforms AA, AB and BB had dose-dependent and divergent effects on spontaneous and cytokine-dependent AML blast proliferation, whereas for constitutive cytokine secretion (IL-1beta, IL-6, TNF-alpha) inhibitory effects were rare and all three isoforms usually had no effect or enhanced the constitutive secretion. The PDGF effects were caused by a direct effect on the AML blasts and were not dependent on the presence of serum. The PDGF effects could also be detected after in vitro culture of AML cells in the presence of IL-4+granulocyte-macrophage colony stimulating factor. PF-4 had divergent effects on proliferation and cytokine secretion by native AML blasts. Our results suggest that exogenous (e.g. platelet-secreted) PDGF and PF-4 can function as regulators of leukemic hematopoiesis and possibly also modulate the function of residual AML cells in peripheral blood stem cell grafts. On the other hand, endogenous release of PDGF-AB by native blasts may modulate the function of normal cells in the bone marrow microenvironment (e.g. bone marrow stromal cells).

Serum levels of thrombopoietin and stem cell factor in acute leukemia patients with chemotherapy-induced cytopenia and complicating infections.

Thrombopoietin (Tpo) and stem cell factor (SCF) are growth factors for megakaryocyte progenitor cells and can also modulate platelet function. We have characterized variations in serum levels of these two cytokines in acute leukemia patients undergoing intensive chemotherapy. Compared with healthy controls, serum Tpo levels were significantly increased prior to consolidation chemotherapy, and serum levels were correlated to peripheral blood platelet counts. Serum Tpo levels increased when the patients developed chemotherapy-induced cytopenia, and a further increase was observed during complicating bacterial infections. In contrast to Tpo, SCF serum levels in leukemia patients did not differ from healthy controls neither before chemotherapy nor during the period of chemotherapy-induced cytopenia. Serum levels of Tpo (and possibly SCF) may influence thrombopoiesis and/or platelet functions in patients undergoing intensive chemotherapy for acute leukemia.

Effects of acute myelogenous leukemia blasts on platelet release of soluble P-selectin and platelet-derived growth factor.

Complex interactions occur between platelets and normal as well as leukemic myeloid cells. In vitro co-culture of platelets and acute myelogenous leukemia (AML) blasts with allogeneic platelets enhances blast proliferation and constitutive cytokine secretion. In the present study the effects of AML blasts on the platelet release of soluble mediators are characterized. Normal platelets released soluble (s) P-selectin and platelet-derived growth factor (PDGF), both when cultured alone and in the presence of AML blasts, and for certain patients the presence of AML blasts increased the platelet release of these mediators. Addition of exogenous interleukin (IL) 10 to platelet-AML blast cultures further increased platelet release of PDGF and sP-selectin. For certain patients decreased AML blast cytokine secretion was observed when PDGF-specific antibodies were added to cultures with blasts plus platelets, these results indicate that platelet release of PDGF is a molecular mechanism for the enhancement of AML blast cytokine secretion. We conclude that complex functional alterations are induced both in AML blasts and normal allogeneic platelets during in vitro co-culture of leukemia cells and platelets.

New strategies in the treatment of acute myelogenous leukemia: mobilization and transplantation of autologous peripheral blood stem cells in adult patients.

During the last decade high-dose Ara-C (HIDAC; single doses of 3 g/m(2)) and autologous stem cell transplantation have been increasingly used as postremission therapy in adult acute myelogenous leukemia (AML). Controlled clinical trials have demonstrated a long-term disease-free survival of 40%-50% for patients treated with at least two courses of HIDAC. Other studies have demonstrated that postremission autologous bone marrow transplantation results in a disease-free survival equal to or better than conventional chemotherapy. However, autotransplantation with mobilized peripheral blood stem cells (PBSC) would now be preferred instead of autologous bone marrow, due to the shorter hematopoietic reconstitution period. The results reviewed in the present article suggest that HIDAC and autologous PBSC transplantation can be combined in the postremission treatment of adult AML, and this combination therapy may also reduce minimal residual disease and the risk of posttransplant relapse. From the available studies it cannot be concluded whether graft purging further reduces the relapse risk. However, the possible advantage of combination therapy with repeated courses of HIDAC and autologous PBSC transplantation needs to be demonstrated in prospective clinical trials before it can be recommended as a part of the routine treatment in AML.

Autologous stem cell transplantation as post-remission therapy in adult acute myelogenous leukemia: does platelet contamination of peripheral blood mobilized stem cell grafts influence the risk of leukemia relapse?

Conventional chemotherapy of acute myelogenous leukemia (AML) results in an overall long-term disease-free survival of less than 50%, but for selected subsets of younger patients the prognosis can be improved by allogeneic stem cell transplantation. The use of autologous stem cell transplantation is now investigated as an alternative to allotransplantation due to its lower risk of serious complications. However, autotransplantation is associated with a relatively high risk of post-transplant AML relapse that can be derived from contaminating leukemia cells in the autograft. Peripheral blood mobilized stem cell (PBSC) grafts usually contain a higher number of platelets. The degree of platelet contamination is determined by the peripheral blood platelet count at the time of harvesting, and the platelets become activated and release soluble mediators during the ex vivo handling of PBSC grafts. Many of these platelet-derived mediators can bind to specific receptors expressed by AML blasts, and the platelet contamination may then alter AML blast survival and thereby influence the risk of post-transplant leukemia relapse. Therefore, we conclude that the platelet contamination of autologous stem cell grafts is possibly of clinical importance, but the effect of this nonstandardized parameter is difficult to predict in individual patients because the number of graft-contaminating platelets, the degree of platelet activation, and the effects of platelet-derived mediators on AML blasts differ between patients.

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts: studies of allostimulated interferon-gamma secretion.

Normal peripheral blood mononuclear cells (PBMC responders) were cultured together with non-irradiated allogeneic PBMC (more than 95% leukaemia blasts) derived from patients with acute leukaemia (referred to as leukaemic PBMC stimulators). Cytokine secretion was determined as cytokine concentrations in supernatants. Both normal PBMC and enriched CD4+ and CD8+ T cells responded to allostimulation with interferon (IFN gamma) secretion. Interleukin-I (IL-1) receptor antagonist and IL-2-neutralizing antibodies decreased IFN gamma secretion. Exogenous IL-1 beta, IL-2 and IL-7 increased allostimulated IFN gamma secretion, whereas decreased levels were seen in the presence of IL-6, IL-10 and granulocyte-colony-stimulating factor (G-CSF). During allorecognition IFN gamma-neutralizing antibodies decreased acute myelogenous leukaemia (AML) blast secretion of G-CSF. We conclude that (i) both CD4+ and CD8+ T cells show allostimulated cytokine secretion in response to allogeneic stimulator cells containing a dominating population of native, cytokine-secreting leukaemia blasts, and (ii) IFN gamma released during this response can modulate the function of allogeneic AML blasts.

New strategies in the treatment of acute myelogenous leukemia (AML): in vitro culture of aml cells--the present use in experimental studies and the possible importance for future therapeutic approaches.

In vitro studies of cultured native acute myelogenous leukemia (AML) blasts and cell lines have contributed significantly to our present knowledge about the pathogenesis of AML. In the present article we review different techniques for preparation and in vitro culture of AML blasts. Well-characterized serum-free in vitro conditions can now be used in experimental studies of AML, and this makes comparisons between different studies easier. We also describe assays for characterization of AML progenitor subsets (i.e., suspension cultures, colony assays, long-term in vitro culture, xenotransplantation in immunocompromised mice), and we discuss the possible use of AML cell lines as experimental models in AML. Furthermore, clinical studies suggest that the in vitro growth characteristics of AML blasts assayed by short-term culture of the total native populations can be used as a predictor of prognosis after intensive chemotherapy. These in vitro assays may therefore be used for more accurate identification of prognostic parameters and thereby form a basis for the development of simplified laboratory techniques suitable for routine evaluation of patients undergoing risk-adapted therapy. However, it will be equally important to further evaluate the clinical relevance of assays for primitive AML progenitors, and to develop simplified methods that can be used to characterize these progenitor subsets in the routine clinical evaluation.

Effects of prostaglandins E1 and E2 on the in vitro production of immunoglobulin by human peripheral blood lymphocytes.

The effects of PGE2 and PGE1 on the response of human peripheral blood lymphocytes to Pokeweed mitogen were studied. Addition of PGE2 inhibited IgM production. This effect was augmented by treating the lymphocytes with 2.0 mug/ml of Indomethacin. Addition of PGE1 alone had little effect but augmentation of IgM production was seen in cultures where the lymphocytes had been treated with Indomethacin and PGE1 then added. The results suggest that PGE1/E2 have a small but measurable effect on in vitro IgM production.

Use of an automated pharmacy system and patient registries to recruit HMO enrollees for an influenza campaign.

OBJECTIVE: To develop methods to identify, recruit, and vaccinate HMO enrollees at increased risk for influenza-related complications as part of a comprehensive influenza campaign. SETTING: Group Health Cooperative (GHC) is a large, membership-governed, managed care organization that serves 395,000 members in the Puget Sound area. APPROACH: An automated pharmacy system and patient data registries were used to identify enrollees with chronic illness. Enrollees with chronic illness and all enrollees 65 years of age and older were considered "high-risk" enrollees to be recruited for vaccination. Postcard reminders coupled with a publicity and education campaign were used to recruit high-risk enrollees to special influenza clinics. RESULTS: Our approach identified 2.5% of children (persons < 18 years of age) and 10.5% of adults (persons 18 to 65 years of age) as chronically ill and thus at high risk for influenza-related complications. Most high-risk children were identified through prescriptions for steroids, autonomic inhalers, or both or because they were enrolled in the asthma registry. Most high-risk adults were identified because of prescriptions for steroids, insulin, or oral hypoglycemic agents; because they had received pneumococcal vaccine; or because they were enrolled in the diabetes registry. Influenza coverage rates for all seniors (persons > or = 65 years of age) increased from 34% in 1984 to more than 72% in the 1996-1997 campaign year. Coverage rates were much lower for high-risk children (36%) and adults (46%). CONCLUSIONS: Influenza coverage rates can still be substantially improved for adults younger than 65 years of age and children at high risk for influenza-related complications.

Resistance of the pulmonary epithelium to movement of buffer ions.

Exposure of the apical surfaces of alveolar monolayers to acidic and alkaline solutions has been reported to have little influence on intracellular pH compared with basolateral challenges (Joseph D, Tirmizi O, Zhang X, Crandall ED, and Lubman RL. Am J Physiol Lung Cell Mol Physiol 282: L675-L683, 2002). We have used fluorescent pH indicators and a trifurcated optical bundle to determine whether the apical surfaces are less permeable to ionized buffers than the membranes that separate the vasculature from the tissues in intact rat lungs. In the first set of experiments, the air spaces were filled with perfusate containing FITC-dextran (mol wt 60000) or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Air space pH fell progressively from 7.4 to 6.61 +/- 0.03 (mean +/- SE, n = 11, air space buffers at 10 mM). Perfusion for 2 min with 2 mM NH4Cl increased air space pH by 0.142 +/- 0.019 unit, without a subsequent acidic overshoot. Infusions of NaHCO3 and sodium acetate reduced pH without a subsequent alkaline overshoot. In the second set of experiments, cellular pH was monitored in air-filled lungs after perfusion with BCECFAM. Injections of NH4Cl caused a biphasic response, with initial alkalinization of the cellular compartment followed by acidification after the NH4Cl was washed from the lungs. Subsequent return of pH to normal was slowed by infusions of 1.0 mM dimethyl amiloride. These studies suggest that lung cells are protected from air space acidification by the impermeability of the apical membranes to buffer ions and that the cells extrude excess H+ through basolateral Na+/H+ exchangers.