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.     

Effects of angiogenic regulators on in vitro proliferation and cytokine secretion by native human acute myelogenous leukemia blasts

Angiogenesis seems to be important in the pathogenesis of acute myelogenous leukemia (AML). The endothelial cell proliferation and microvessel formation are regulated by a wide range of soluble mediators, including angiogenin, angiopoietin-2, basic fibroblast growth factors, vascular endothelial growth factor (VEGF), VEGF-D, angiostatin and endostatin. In the present study, it has been investigated whether these mediators have an additional direct effect on the proliferation and cytokine release by native human AML blasts. AML cells derived from a large group of consecutive patients were investigated. All these mediators could alter the proliferation and cytokine release [interleukin (IL) 1beta, IL6, IL8, tumor necrosis factor alpha] for a minority of patients. Alteration of spontaneous proliferation by at least one mediator was detected in five of 38 patients; whereas, altered cytokine (Flt3-ligand, granulocyte-macrophage colony-stimulating factor, stem cell factor)-dependant proliferation was observed for 10 patients. Growth enhancement was most frequently observed, whereas growth inhibition was uncommon. The effects on AML blast proliferation were often dependant on or were modulated by the presence of the three hematopoietic growth factors. Based on the present results, it is concluded that angioregulatory mediators have additional growth-enhancing effects directly on the AML blasts for certain patients. However, based on the results from this investigation and previous studies it is suggested that their major contribution to the pathogenesis of AML is through their effects on regulation of bone marrow angiogenesis, and future studies of these mediators in AML should probably focus on these effects.     

In vitro effects of insulin-like growth factor-1 (IGF-1) on proliferation and constitutive cytokine secretion by acute myelogenous leukemia blasts

The effects of insulin-like growth factor 1 (IGF-1) on autonomous proliferation, cytokine-dependent proliferation and constitutive cytokine secretion by acute myelogenous leukemia (AML) blasts were investigated using serum-free in vitro conditions. IGF-1 enhanced AML blast proliferation independent of the presence of other exogenous cytokines only for 2 of 21 patients, but for 10 additional patients IGF-1 altered blast proliferation in the presence of certain exogenous cytokines or cytokine combinations. IGF-1 had minor effects on AML blast cytokine secretion only for a subset of the patients (decreased levels for 1 patient, increased levels for 7 patients). Our in vitro observations indicate that IGF-1 can modulate AML blast proliferation and/or cytokine secretion for a subset of patients.     

Effect of thrombopoietin on proliferation of blasts from CD7-positive acute myelogenous leukaemia

We investigated the effect of thrombopoietin (TPO) on the growth of leukaemic blasts from 30 acute myelogenous leukaemia (AML) patients according to the surface expression of CD7 and CD34: 10 patients were CD7 positive (CD7⁺), nine were CD7 negative/CD34⁺(CD7^?/CD34⁺) and the remaining 11 were CD7^?/CD34^?. Significant growth response of leukaemic blasts to TPO was observed in 10/10 CD7⁺, 5/9 CD7^?/CD34⁺ and 2/11 CD7^?/CD34^? AML cases using ³H-thymidine incorporation. Synergistic stimulatory effects of TPO with stem cell factor (SCF), interleukin-3 (IL-3), granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor were observed in both TPO-responding cases (9/17) and TPO-non-responding cases (8/13). In a leukaemic blast colony assay, significant growth response to TPO was observed in 5/6 CD7⁺ and 4/17 CD7^? AML cases examined. However, the effect of TPO on the growth of CD7⁺ leukaemic blasts was not so potent as that of IL-3 and SCF, both of which support the proliferation of primitive haemopoietic progenitors. Expression of c-mpl (TPO receptor) was significantly higher in CD7⁺ AML cases than in CD7^? cases, suggesting a relationship between expression of c-mpl and proliferative response to TPO. These data indicate that CD7⁺ leukaemic blasts express functional TPO receptors and proliferate in response to TPO. These observations also imply that CD7 expression on AML blasts may indicate involvement of leukaemic progenitors at an early stage of multipotent haemopoietic stem cells.     

Variable differentiation patterns of acute myelogenous leukaemia blasts in liquid suspension cultures

To study the ability of acute myelogenous leukaemia blasts to spontaneously differentiate in vitro, bone marrow and/or blood mononuclear cells from 63 patients with acute myelogenous leukaemia were incubated in liquid suspension cultures containing human serum, without addition of chemical inducers of differentiation. Cultures were examined weekly for disappearance or persistence of blasts, and for appearance of morphologically recognizable granulocytes and macrophages. Culture outcomes were extremely variable, ranging from lack of appearance of differentiated cells to complete disappearance of blasts with replacement by mature cells. In 50 cases an increase (25-185%) in the absolute number of differentiated cells in culture was noted during the culture period. Full differentiation was seen exclusively in cultures from 13/48 (27.1%) patients studied at diagnosis, as compared to 0/20 patients studied at relapse (P less than 0.01). The ability to fully or partially differentiate in culture was lost to a significant degree at relapse (13/20 patients) as compared to diagnosis (48/48 patients, P = 0.0001). At diagnosis full differentiation in culture was associated with a significantly higher remission rate than partial differentiation (89% versus 40%, P less than 0.02). Origination of mature cells from leukaemic rather than normal precursors was suggested by the appearance of Auer rods in mature cells in seven cases, by the correlation of types of differentiated cells seen in culture with the FAB class of leukaemia and by cytogenetic data in one case.     

Unstimulated human acute myelogenous leukemia blasts secrete matrix metalloproteinases

Purpose: The secretion of metalloproteinases was examined, especially the 92-kDa and 72-kDa type IV collagenases/gelatinases, and their role in the degradation of reconstituted basement membrane (Matrigel) by leukemic blasts.Methods and results: Leukemic blasts were obtained from the peripheral blood of 11 patients diagnosed with acute myelogenous leukemia (AML). After incubation of the AML blasts in serumfree cultures, conditioned media were collected and examined by zymography. The 92-kDa gelatinase was detected in ten AML patients after 2 h and 24 h of incubation, and in five samples its activated form (83 kDa) was observed. 72-kDa gelatinase was detected in cell-conditioned media from four patients after 2 h and in media from ten patients after 24 h. Its activated forms (64–60 kDa) were observed in one of four samples after 2 h and in five of ten after 24 h. By contrast, normal peripheral mononuclear cells from healthy donors secreted only 92-kDa gelatinase after 24 h; the 72 kDa enzyme was not detectable. A specific inhibitor of metalloproteinases, 1, 10-phenanthroline, significantly reduced the in vitro invasion of AML blasts in a Matrigel assay and completely inhibited gelatinolytic activity in zymography.Conclusions: We concluded that primary, unstimulated peripheral-blood AML blasts secrete metalloproteinases, which may contribute to the in vitro degradation of reconstituted basement membrane.Abbreviations AML acute myelogenous leukemia - MMP matrix-degrading metalloproteinase - MNC mononuclear cells - IMDM Iscove's modified Dulbecco's medium - BSA bovine serum albumin     

Effects of nonpathogenic bacteria on cytokine secretion by human intestinal mucosa

OBJECTIVE: The human intestine harbors a complex microbial ecosystem, and the mucosa is the interface between the immune system and the luminal environment. The aim of this study was to elucidate whether host-bacteria interactions influence mucosal cytokine production. METHODS: Macroscopically normal colonic specimens were obtained at surgery from eight patients with neoplasm, and inflamed ileal specimens were obtained from two patients with Crohn's disease. Mucosal explants were cultured for 24 h with either nonpathogenic Escherichia coli ECOR-26, Lactobacillus casei DN-114 001, L. casei DN-114 056, L. casei ATCC-334, or Lactobacillus bulgaricus LB-10. Each study included blank wells with no bacteria. Tissue and bacteria viability were confirmed by LDH release and culture. Concentration of tumor necrosis factor (TNF)alpha, transforming growth factor beta1, interleukin (IL)-8, and IL-10 was measured in supernatants. In parallel experiments, neutralizing anti-TNFalpha antibody was added to the culture. RESULTS: Co-culture of mucosa with bacteria did not modify LDH release. Co-culture with L. casei strains significantly reduced TNFalpha release, whereas E. coli increased it. These effects were observed both in normal and inflamed mucosa. In combination studies, L. casei DN-114 001 prevented TNFalpha stimulation by E. coli. L. casei DN-114 001 also reduced IL-8 release via a TNFalpha-independent pathway. L. casei DN-114 056 or E. coli increased IL-10 release in the presence of neutralizing anti-TNFalpha. CONCLUSIONS: Nonpathogenic bacteria interact with human intestinal mucosa and can induce changes in cytokine production that are strain specific.     

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.     

Effects of interleukin-4 and interleukin-6 on the proliferation of CD34+ and CD34- blasts from acute myelogenous leukemia.

We studied the effects of interleukin-4 (IL-4) and IL-6 on the growth of leukemic blasts from 40 patients with acute myelogenous leukemia (AML). Patients were selected on the basis of negativity for a series of B-cell antigens including CD10 and CD19. Twenty-one cases were CD34-positive (CD34+) (greater than 15% of blasts) and the remaining 19 were CD34-negative (CD34-) (less than 3% of blasts). IL-4 alone (100 U/ml) could stimulate either DNA synthesis (with greater than 2.0 stimulation index) or leukemic blast colony formation in 24 of 40 AML patients. In the presence of other growth factors, IL-4 showed divergent effects on IL-3-, granulocyte-macrophage colony-stimulating factor-, granulocyte colony-stimulating factor-, or erythropoietin-dependent colony formation. These effects of IL-4 were observed in both CD34+ and CD34- AML cases. IL-6 (100 U/mL) alone could not stimulate DNA synthesis and blast colony formation except for one CD34+ case. On the other hand, IL-6 showed synergistic effects on IL-3- and IL-4-dependent blast colony formation in 10 of 12 and 7 of 9 CD34+ AML cases, respectively. Among CD34- AML cases, such synergism was seen only in 1 of 12 cases for IL-3-dependent colony formation and in 3 of 7 cases for IL-4-dependent colony formation. The divergent effect of IL-4 and the synergistic effect of IL-6 were also observed in purified CD34+ leukemic blast populations, indicating that these phenomena are not mediated by accessory cells. The present study suggests that IL-4, alone or in combination with other growth factors, has divergent effects on the growth of AML progenitors irrespective of the CD34 expression, and that IL-6 acts synergistically with IL-3 or IL-4 on the growth of leukemic progenitors preferentially in CD34+ AML.     

Expression of human leucocyte antigens and co-stimulatory molecules on blasts of patients with acute myeloid leukaemia

Recently, leukaemia-associated antigens (LAA) recognized by T lymphocytes, such as Wilm's tumour-1 (WT-1) or pathogenesis-related protein-1 (PR-1), have been identified. For immunotherapies that employ antigen peptides, either alone or pulsed on dendritic cells (DC), the expression of human leucocyte antigen (HLA) molecules on the targeted leukaemic blasts (LB) is crucial. The co-stimulatory molecules CD80 and CD86 give the secondary signal to T lymphocytes that is necessary for the lysis of leukaemia cells, and CD40 enhances the efficacy of antigen presentation. Here, the expression of HLA-ABC, HLA-A2, HLA-DR, CD40, CD80 and CD86 was flow cytometrically examined in blood samples from 24 healthy volunteers (HV), 24 patients with newly diagnosed acute myeloid leukaemia (AML) and five patients with relapsed AML. The expression of HLA-ABC, CD40, CD80 and CD86 was significantly reduced on LB in comparison with monocytes of HV. HLA-A2 and HLA-DR expression was similar on LB and on monocytes of HV. In AML patients, the expression of HLA and CD86 molecules was significantly higher on LB than on CD33/CD34-negative monocytes. CD40 and CD80 molecules were deficient on AML blasts. The preservation of HLA molecules and CD86 on LB of the majority of AML patients at the time of diagnosis and even at relapse of the disease are prerequisites for LAA-targeted immunotherapies in these patients.     

Lipoteichoic acid derived from Enterococcus faecalis modulates the functional characteristics of both normal peripheral blood leukocytes and native human acute myelogenous leukemia blasts

OBJECTIVES: Several case reports have described complete hematological remissions for patients with otherwise untreated acute myelogenous leukemia (AML) who receive hematopoietic growth factor therapy during complicating bacterial infections. This may be caused by indirect cytokine effects, but direct effects of infecting agents on the malignant cells are also possible because bacterial molecules can bind to specific receptors expressed by normal and malignant leukocytes. Lipoteichoic acid (LTA) is a cell wall component of gram-positive bacteria, and it can activate normal immunocompetent cells through binding to specific cell membrane receptors. METHODS: We investigated effects of LTA derived from Enterococcus faecalis on in vitro cultured (i) normal peripheral blood mononuclear cells (PBMC); (ii) remaining T cells derived from patients with hematologic malignancies and chemotherapy-induced leukopenia; and (iii) native human AML cells. RESULTS: Increased interleukin 1beta (IL1beta) and IL8 release by in vitro cultured normal PBMC was observed after stimulation with LTA at concentrations > or =5 microg/mL; these levels were lower than for lipopolysaccharide (LPS)-stimulated cells and LTA antagonized LPS-induced cytokine release by normal PBMC. In most cases LTA did not alter T-cell proliferation for patients with chemotherapy-induced leukopenia. The LTA effects on AML blasts were investigated for 62 consecutive patients. LTA altered either cytokine (granulocyte-macrophage colony-stimulating factor + stem cell factor + IL3)-dependent proliferation or the release of IL1beta/IL8 for 23 patients; the effects were divergent but increased proliferation/cytokine levels were most commonly observed. CONCLUSION: The LTA derived from E. faecalis can modulate the functional characteristics of normal leukocytes and native human AML blasts.     

Effects of homo-aza-steroids on acute non-lymphocytic leukaemia cell proliferation in vitro

Summary. Homo-aza-steroids (modified steroid molecules) in their esterified forms have been used extensively as carrier molecules of alkylating agents against several neoplastic malignancies in vivo and in vitro. We studied the effects of two homo-aza-steroid carrier molecules alone, namely 3β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13, 17-lactam (compound 1) and 13α-amino-13,17-seco-1,3,5-estratrien-17-oic-13,17-lactam (compound 2), on human acute non-lymphocytic leukaemia cell proliferation in vitro.
We used peripheral blood samples from 27 untreated ANLL patients (eight M1, four M2, two M3, six M4, three M5a, two M5b and two M6, according to FAB criteria). Proliferative activity was estimated by using thymidine uptake and the percentage of cells in metaphase in 24, 48 and 72 h of culture. Exposure of human leukaemic blasts with either of the two compounds resulted in enhanced cell proliferation in M1, M2, M4, M6 and M5a (only by compound 2) cases, whereas there was no significant effect in the M3 and M5b cases.
Our results indicate that the two compounds tested exhibit stimulatory effect on cell proliferation, particularly in blast cells possessing a relatively smaller degree of differentiation (Ml and M6 cases exhibiting CD34 and CD7). Further research is needed to study the cell growth effect and the therapeutic potential of these steroid molecules in human blood malignancies in vitro and in vivo.     

Effects of dexamethasone on proliferation, activity, and cytokine secretion of normal human bone marrow stromal cells: possible mechanisms of glucocorticoid-induced bone loss.

It is well documented that glucocorticoid excess causes bone loss, but the mechanisms of these effects remain poorly defined. To understand further the mechanisms of glucocorticoid-induced osteoporosis, we investigated the effects of glucocorticoids on bone formation and bone resorption by examining the proliferation, functional activities, and cytokine secretion of cultured human bone marrow stromal cells (hBMSC). Treatment with dexamethasone for 24 h at the concentration of 10(-8) M significantly suppressed [(3)H]thymidine incorporation and further inhibition was observed with longer treatment (8 days) or higher concentration (10(-7) M). Alkaline phosphatase activity of hBMSC was markedly stimulated with addition of dexamethasone (10(-8) M), to 191 +/- 22% (after 4 days) and 317 +/- 46% (after 7 days) of control. Dexamethasone (10(-8) M) treatment for 48 h decreased the incorporation of [(3)H]proline into collagenase-digestible protein (CDP; 43.7+/-7.9% of control) and non-collagen protein (65.2+/-8.4% of control), with a greater effect on CDP. Northern blot analysis indicated that alpha1(I)-collagen mRNA level was decreased by dexamethasone to 27.6 +/- 9.0% of the control value after 1 day of exposure, and to 55.2 +/- 6.2% after 7 days. Dexamethasone markedly suppressed basal production of interleukin (IL)-6 and IL-11 and that stimulated by parathyroid hormone (PTH), IL-1alpha, or tumour necrosis factor-alpha in a dose-dependent manner. These results suggest that the glucocorticoid-induced bone loss is derived at least in part via inhibition of bone formation, which includes the suppression of osteoblast proliferation and collagen synthesis. As both basal and PTH-stimulated production of IL-6 and IL-11 are decreased by dexamethasone, the increased bone resorption observed in glucocorticoid-induced osteopenia does not appear to be mediated by IL-6 or IL-11.     

How to address second and therapy-related acute myelogenous leukaemia

Secondary acute myelogenous leukaemia (AML), as compared to de novo AML, occurs in the more elderly population, is independently more resistant to cytotoxic chemotherapy, has a higher relapse rate, and a worse prognosis. Secondary AML (sAML) is a heterogeneous disease, both biologically and clinically, even within the World Health Organization subgroups of sAML. Outcomes are the poorest in subgroups with sAML arising from an antecedent haematologic disorder which has been previously treated (ts-AML), and sAML in patients <55 years of age. This review describes the suboptimal outcomes of contemporary therapy, to support the notion of an unmet need for innovative treatment strategies in sAML. Despite the recent approval of CPX-351, long-term outcomes for this high-risk disease remain dismal. Resistance mechanisms to intensive chemotherapy contribute to relapse. Targeted immune therapy may avoid multidrug resistance mechanisms, but are unlikely to provide long-term remission due to a complex and rapidly evolving clonal disease profile. Advances for sAML will likely be accomplished by CAR T cell therapy or bispecific antibodies providing a bridge to allogeneic stem cell transplantation. Therefore, focus should be placed on novel strategies that can augment the untargeted effector function of allogeneic grafts.