A pilot study of single nucleotide polymorphisms in the interleukin-6 receptor and their effects on pre- and post-transplant serum mediator level and outcome after allogeneic stem cell transplantation.

Interleukin (IL)-6 is an important regulator of immunity and inflammation in many diseases. Single nucleotide polymorphisms (SNPs) in the IL-6 gene influence outcome after allogeneic stem cell transplantation (ASCT), but the possible importance of SNPs in the IL-6 receptor has not been examined. We therefore investigated whether SNPs in the IL-6R gene influenced biochemical characteristics and clinical outcomes after ASCT. We examined the IL-6 promoter variant rs1800975 and the IL-6R SNPs rs4453032, rs2228145, rs4129267, rs4845374, rs4329505, rs4845617, rs12083537, rs4845618, rs6698040 and rs4379670 in a 101 population-based cohort of allotransplant recipients and their family donors. Patients being homozygous for the major alleles of the IL-6R SNPs rs2228145 and rs4845618 showed high pretransplant CRP serum levels together with decreased sIL-6R levels; the decreased IL-6R levels persisted 6 months post-transplant. In contrast, patients being homozygous for the minor allele of the IL-6R SNP rs4379670 showed decreased pretransplant CRP levels. Furthermore, the IL-6R rs4845618 donor genotype showed an association with severe acute graft-versus-host disease (GVHD), whereas the donor genotype of the IL-6 SNP rs1800795 was associated with decreased survival 100 days post-transplant. Finally, the recipient genotype of the IL-6R SNP rs4329505 showed a strong association with 2-years non-relapse mortality, and this effect was also highly significant in multivariate analysis. IL-6 and IL-6R SNPs influence the clinical outcome after allogeneic stem cell transplantation.

A prospective observational study on effects of fever on red cell transfusion outcome.

The effects of fever on red cell transfusions are not well documented. In this pilot study, we have compared the outcome of red-blood-cell transfusions in haematologic patients with and without fever. The results indicate that haemoglobin increment per unit is significantly lower in febrile patients receiving red cell transfusions than in patients without fever. These findings are in line with earlier findings in preclinical studies. Larger studies are necessary to confirm our results, and laboratory studies should be conducted to investigate the underlying mechanisms.

Hyperleukocytosis and leukocytapheresis in acute leukaemias: experience from a single centre and review of the literature of leukocytapheresis in acute myeloid leukaemia.

BACKGROUND: Hyperleukocytosis is usually defined as leukocyte count >100 × 10(9) L(-1) and can be seen in newly diagnosed leukaemias. Hyperleukocytic leukaemia is associated with a risk of organ failure and early death secondary to leukostasis. Mechanical removal of leukocytes by the apheresis technique, leukocytapheresis, is a therapeutic option in these patients. METHODS: During a 16-year period, 16 patients were treated with leukocytapheresis (35 apheresis procedures) for hyperleukocytosis/leukostasis. We present our experience, and in addition we review previous studies of hyperleukocytosis/leukocytapheresis in patients with acute myeloid leukaemia (AML). RESULTS: We used a highly standardised approach for leukocytapheresis in leukaemia patients with hyperleukocytosis. The average leukocytapheresis number for each patient was 2·2 (range 1-6). Median leukocyte count before apheresis was 309 × 10(9) L(-1) (range 104-935); the mean leukocyte count reduction was 71%, corresponding to a mean absolute reduction of 219 × 10(9) L(-1). No serious side effects were seen during or immediately after apheresis. CONCLUSIONS: The data suggest that our standardised technique for leukocytapheresis effectively reduced the peripheral blood leukaemia cell counts. Previous studies in AML also support the conclusion that this is a safe and effective procedure for the treatment of a potentially life-threatening complication, but apheresis should always be combined with early chemotherapy.

The chemokine system and its contribution to leukemogenesis and treatment responsiveness in patients with acute myeloid leukemia.

The chemokine family consists of approximately 50 small (8-14 kDa), basic proteins that are expressed and released by a wide range of normal and malignant cells. Most chemokines act through heptahelical transmembrane G protein- coupled receptors. Based on their molecular structure these cytokines are divided into the two major subgroups CCL and CXCL chemokines that bind to CCR or CXCR receptors respectively. Primary human acute myelogenous leukemia (AML) cells show constitutive release of a wide range of chemokines, but the chemokine release profile differs between patients. Among the commonly expressed chemokines are proangiogenic CXCL8, antiangiogenic CXCL4/9-11 and several leukocyte-chemotactic chemokines. Systemic serum levels of leukocyte-chemotactic chemokines depend both on patient age, disease status, the chemotherapy regimen and development of complicating infections. The local chemokine network in human AML is probably further modulated by the hypoxic bone marrow microenvironment and the local release of chemokines by nonleukemic bone marrow stromal cells. Usually primary AML cells also express several chemokine receptors. Specific chemokine inhibitors are now being developed, including chemokine-neutralizing or receptor-blocking antibodies, antisense strategies, receptor-blocking small molecules or inhibitors of downstream signaling. The use of CXCR4-antagonists for mobilization of peripheral blood stem cells has been documented in several clinical studies. Although animal studies suggest that chemokine inhibition also may become useful in the treatment of graft versus host disease, the possible use of chemokine-targeting therapy for other indications than stem cell mobilization requires further studies.

Animal models of acute myelogenous leukaemia - development, application and future perspectives.

From the early inception of the transplant models through to contemporary genetic and xenograft models, evolution of murine leukaemic model systems have been critical to our general comprehension and treatment of cancer, and, more specifically, disease states such as acute myelogenous leukaemia (AML). However, even with modern advances in therapeutics and molecular diagnostics, the majority of AML patients die from their disease. Thus, in the absence of definitive in vitro models which precisely recapitulate the in vivo setting of human AMLs and failure of significant numbers of new drugs late in clinical trials, it is essential that murine AML models are developed to exploit more specific, targeted therapeutics. While various model systems are described and discussed in the literature from initial transplant models such as BNML and spontaneous murine leukaemia virus models, to the more definitive genetic and clinically significant NOD/SCID xenograft models, there exists no single compendium which directly assesses, reviews or compares the relevance of these models. Thus, the function of this article is to provide clinicians and experimentalists a chronological, comprehensive appraisal of all AML model systems, critical discussion on the elucidation of their roles in our understanding of AML and consideration to their efficacy in the development of AML chemotherapeutics.

Acute myelogenous leukemia blasts as accessory cells during T lymphocyte activation: possible implications for future therapeutic strategies.

Both clinical and experimental evidence indicate that T lymphocytes can mediate antileukemic effects in acute myelogenous leukemia (AML). These antileukemic effects can be either nonspecific cytotoxicity (killer cell activity) or reactivity against leukemia-specific antigenic peptides presented by self-HLA molecules. The antigen-specific T cell activation requires recognition of specific peptides together with costimulatory signalling. For most patients the AML blasts express both HLA class I and class II molecules for antigenic presentation, but patients are heterogeneous with regard to: (1) expression of costimulatory binding molecules; (2) expression of receptors/counterreceptors involved in induction of apoptosis; (3) constitutive release of immunomodulatory soluble mediators. This heterogeneity suggests that the ability of AML blasts to initiate an antileukemic T cell response will differ between individual patients. Thus, clinical approaches for immunotherapy in AML have to overcome three major problems. First, the therapy should reduce the patient heterogeneity so that therapeutic effects become more predictable; or alternatively one should define patient subsets which are likely to benefit from immunotherapy. Second, immunotherapy should enhance antileukemic T cell reactivity or blast susceptibility to immune attacks. Third, the therapeutic procedures must be safe and suitable for routine use. All three problems probably have to be solved before immunotherapy can become a routine treatment.

Effects of interleukin-13 on cytokine secretion by human acute myelogenous leukemia blasts.

Blast cells derived from patients with acute myelogenous leukemia (AML) were cultured in the presence of interleukin-13 (IL-13). IL-13 did not cause statistically significant alterations of AML blast proliferation when cells were cultured in medium alone or together with IL-4, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. In contrast, IL-13 inhibited constitutive AML blast secretion of IL-1 alpha, IL-1 beta, IL-6, tumor necrosis factor alpha, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. IL-4 caused a similar inhibition of constitutive cytokine secretion as IL-13, but IL-13 caused no additive inhibition in the presence of IL-4. In contrast to IL-4 which increased AML blast release of IL-1 receptor antagonist, IL-13 caused no significant alteration of blast release of the receptor antagonist. IL-13 inhibited cytokine secretion also in the presence of neutralizing IL-4 and IL-10 antibodies and when AML blasts were cultures in serum-free conditions. We conclude that IL-13 has a direct and nontoxic inhibitory effect on constitutive AML blast cytokine secretion.

Antiproliferative effects of a non-beta-oxidizable fatty acid, tetradecylthioacetic acid, in native human acute myelogenous leukemia blast cultures.

The lipid metabolism is important in the regulation of cell proliferation. We have examined effects of a fatty acid analogue, tetradecylthioacetic acid (TTA), on the functional phenotype of native, human AML cells. TTA inhibited AML blast proliferation in the presence of single cytokines (GM-CSF and SCF: P > 0.05, 35 patients with detectable proliferation) and a combination of cytokines (P < 0.005, n = 21). This antiproliferative effect was generally stronger than for the normal fatty acid palmitic acid (PA). Both TTA and PA increased the secretion of tumor necrosis factor alpha (TNFalpha) (P < 0.05, 27 patients with detectable cytokine release), but only PA increased interleukein 1beta (IL-1beta) release (P < 0.005, n = 34). AML blast populations varied significantly in their levels and activities of metabolites and enzymes characterizing oxidative status and fatty acid metabolism, and there was no significant correlation between the intrinsic oxidative status and the effects of PA and TTA on blast proliferation. Although TTA reduced the proliferation of mitogen-stimulated normal T cells derived from healthy individuals (P < 0.05, n = 8), no adverse effects were seen on peripheral blood cell counts (reticulocytes, platelets, total white blood cells, differential leukocyte counts) for healthy volunteers receiving TTA (oral administration of 1000 mg/day for 7 consecutive days). Our results suggest that TTA can inhibit AML blast proliferation through pathways that are unrelated to autocrine cytokine secretion and intrinsic oxidative status.

Effects of interleukin 10 on blast cells derived from patients with acute myelogenous leukemia.

The effect of interleukin 10 (IL-10) on proliferation and cytokine secretion by acute myelogenous leukemia (AML) blast cells was investigated in vitro. IL-10 inhibited spontaneous AML blast proliferation for a majority of patients, whereas in the presence of exogenous growth factors (granulocyte-stimulating factor, G-CSF; granulocyte-macrophage colony-stimulating factor, GM-CSF; interleukin 3) the IL-10 effect on blast proliferation showed a wide variation depending on the individual AML patient. IL-10 seemed to cause an irreversible inhibitory effect on AML blasts, as inhibition could also be demonstrated when IL-10 was present only during the initial preincubation of the leukemia cells. IL-10 also inhibited AML blast colony formation. However, independent of the effect on AML blast proliferation, IL-10 decrease cytokine secretion from AML blast cells for all patients, as demonstrated for IL-1 alpha, IL-1 beta, tumor necrosis factor-alpha, GM-CSF and interleukin 6. IL-10 did not inhibit development of apoptosis in AML blasts cultured in vitro. Expression of complement receptors and capability to adhere and internalize bacteria by AML blasts were not altered by IL-10.

At least five antigenic epitopes on the streptokinase molecule are recognized by human CD4+ TCR alpha beta+ T cells.

The streptokinase molecule (415 AA) was cleaved at methionine 237, 347 and 370 yielding four polypeptide fragments. Human HLA-class II restricted streptokinase-specific T cell clones and cell lines (CD2+, CD3+, CD4+, CD8-, TCR alpha beta+, TCR gamma delta-) recognized antigenic epitopes on all four fragments AA 1-236, AA 238-346, AA 348-369 and AA 371-415. T cell clones recognizing fragment AA 1-236 were restricted by at least two different HLA-class II elements, this indicating that more than one antigenic epitope can be recognized on this fragment. In addition, two streptokinase-specific T cell clones recognized only the intact molecule and none of the molecular fragments. These two clones probably recognized an antigenic epitope including one of the methionine residues used for molecular cleavage. We conclude that T cell proliferative responses to streptokinase are determined by recognition of at least five different antigenic epitopes distributed along the entire streptokinase polypeptide chain.

Cytokine responsiveness of mitogen-activated T cells derived from acute leukemia patients with chemotherapy-induced leukopenia.

The aim of the study was to characterize effects of exogenous cytokines on T lymphocytes derived from acute leukemia patients with chemotherapy-induced leukopenia. We investigated the cytokine responsiveness of long-term expanded CD4+ and CD8+ T cell clones and the effects of exogenous cytokines on anti-CD3-stimulated polyclonal T cell responses. After mitogenic activation in the presence of acute myelogenous leukemia (AML) blasts, most CD4+ and CD8+ clones proliferated in response to interleukin-2 (IL-2). Although a majority of the IL-2-responsive clones could also proliferate in the presence of exogenous IL-4, IL-7, IL-9, IL-10, IL-12, and IL-15, only IL-15 responses were equal to or exceeded the corresponding IL-2 responses. Exogenous cytokines were also added during T cell activation with phytohemagglutinin (PHA) + accessory leukemia cells derived from different AML patients, and all the cytokines then had divergent effects that depended on both differences between clones and differences between AML patients. However, for most of these T cell clone/AML blast combinations, IL-2 and IL-15 caused enhanced T cell proliferation. IL-2 and IL-15 also enhanced anti-CD3-stimulated polyclonal responses of nonexpanded T cells derived from cytopenic patients, whereas other cytokines had only minor effects. Our results demonstrate that cytokine-responsive T cells remain in the circulation during chemotherapy-induced cytopenia, and combination therapy including intensive chemotherapy and T cell-targeting cytokine therapy should, therefore, be possible in AML.

Production of interleukin 2 containing growth medium for cloning of human T lymphocytes.

Culture conditions which would enhance production of interleukin 2 (IL-2) from PHA-stimulated peripheral blood mononuclear cells (PBM) were studied. One thousand rads. irradiation of the cells was found to alter the kinetics and to increase the production of IL-2. Addition of indomethacin or irradiated EBV-transformed B lymphocytes to the cultures, removal of adherent cells, and 18 h preincubation of the cells before PHA stimulation had an additive enhancing effect on the IL-2 production. Following these studies a simple and efficient method producing IL-2 for cloning of human T cells was developed.

Effects of endogenous interleukin 1 on blast cells derived from acute myelogenous leukemia patients.

The in vitro effects of interleukin 1 (IL1) secreted by acute myelogenous leukemia (AML) blasts (termed endogenous IL 1 secretion) were investigated. Interleukin 1-dependent AML blast functions were inhibited during in vitro culture either by IL1-specific neutralizing antibodies (anti-IL1 alpha and anti-IL1 beta) or by the IL1 receptor antagonist (IL1RA). Endogenous IL1 secretion showed a wide variation between individual patients, but despite this variation IL1 inhibition significantly decreased both spontaneous blast proliferation and spontaneous blast secretion of IL1 alpha, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha and interleukin 6. In contrast to spontaneous blast proliferation, in the presence of exogenous hematopoietic growth factors (granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin 3, tumor necrosis factor alpha, stem cell factor), IL1 inhibition caused either increased or decreased AML blast proliferation depending on the individual patient. When AML blasts were cultured with stem cell factor plus granulocyte-macrophage colony-stimulating factor, IL1 inhibition significantly increased AML blast proliferation. Thus, IL1 is important for regulation of AML blast proliferation and cytokine secretion independent of the level of endogenous IL1 secretion, but the final effect of IL1 is highly dependent on the cytokine network in the AML blast microenvironment.

Effects of dipyridamole and R-verapamil on in vitro proliferation of blast cells from patients with acute myelogenous leukaemia.

Cytokine-dependent AML cell proliferation was investigated in 16 patients. Dipyridamole and R-verapamil caused a dose-dependent inhibition of AML cell proliferation, and for both drugs the degree of inhibition was similar when testing various haematopoietic growth factors or growth factor combinations (IL3, G-CSF, GM-CSF, G-CSF + GM-CSF, TNF-alpha + GM-CSF). TNF-alpha alone increased AML cell proliferation for five patients, whereas four patients showed unaltered or decreased proliferation. Independent of this TNF-alpha effect, R-verapamil inhibited proliferation for all AML patients in the presence of TNF-alpha, whereas dipyridamole caused only a weak inhibition.

Expression and release of adhesion molecules by human acute myelogenous leukemia blasts.

The expression and release of adhesion molecules by acute myelogenous leukemia (AML) blasts was investigated in vitro. For most patients AML blasts expressed relatively low levels of membrane-bound L-selectin and Intercellular adhesion molecule 1 (ICAM-1), but their soluble forms were detected in the supernatants for the majority of patients when AML blasts were cultured in vitro. These in vitro levels of SL-selectin and sICAM-1 were considerably lower than the normal serum levels. Divergent and relatively small alterations in SL-selectin and sICAM-1 levels were usually observed when exogenous growth factors were present during AML blast culture, whereas increased SL-selectin levels were observed after coculture of AML blasts and normal leucocytes. E- and P-selectin were neither expressed nor released by AML blasts. We conclude that AML blasts are a source of soluble adhesion molecules.

Expression of Fc(epsilon)-receptors by human acute myelogenous leukemia (AML) blasts: studies of high- and low- (CD23) affinity receptor expression and the effects of IgE-mediated receptor ligation on functional AML blast characteristics.

Acute myelogenous leukemia (AML) blasts derived from 20 patients were examined for expression of high- (Fc(epsilon)RI) and low-affinity (Fc(epsilon)RII, CD23) IgE Fc(epsilon)-receptors. Fc(epsilon)RI expression was not detected for any patient. In contrast, expression of CD23 (at least 15% of the blasts stained positive) was detected for 6 out of the 20 patients. Acute lymphoblastic leukemia (ALL) blasts derived from 12 patients did not express CD23 (<1% positive cells for all patients). The functional effects of Fc(epsilon)R-receptor ligation were also examined for 20 patients, including the five patients with highest CD23 expression (30-55% positive cells) and five patients with verified low CD23 expression (

Cytokine modulation of interleukin 1 and tumour necrosis factor-alpha secretion from acute myelogenous leukaemia blast cells in vitro.

Spontaneous secretion of interleukin 1 (IL-1) alpha, IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) from acute myelogenous leukaemia (AML) blasts showed significant correlation, and detectable levels of all cytokines were seen for a majority of patients. IL-3 and granulocyte/macrophage colony-stimulating factor increased secretion of IL-1 alpha, IL-1 beta and TNF-alpha for a majority of AML patients, whereas IL-4 decreased cytokine secretion. The effect of IL-6 and stem cell factor on cytokine secretion varied between different patients. A wide variation in IL-1 alpha, IL-1 beta and TNF-alpha secretion between different patients was seen both for spontaneous secretion and in the presence of all cytokines.

Serum concentrations of tumour necrosis factor-alpha during chemotherapy-induced leukopenia in patients with acute leukaemia and bacterial infections.

Serum concentrations of tumour necrosis factor-alpha (TNF-alpha) increase during septicaemia in previously healthy individuals. To investigate whether a similar increase in TNF-alpha can be seen in severely immunocompromised patients with acute leukaemia and chemotherapy-induced leukopenia, serum TNF-alpha was analysed in leukopenic patients with bacterial infections. Pretherapy serum levels of TNF-alpha were decreased in leukaemia patients compared with healthy controls, and serum TNF-alpha levels showed a further decrease when patients developed chemotherapy-induced leukopenia. When leukopenic patients developed bacterial infections, serum concentrations of TNF-alpha increased. Serum levels of TNF-alpha decreased when clinical signs of infection resolved during antibiotic therapy, but an increase occurred later in parallel with haematopoietic reconstitution.

Ribosomal proteins sustain morphology, function and phenotype in acute myeloid leukemia blasts.

Translation of mRNA is a prerequisite for cell proliferation, differentiation and viability. We have studied the effect of ribosome protein factors (GPRE) on acute myeloid leukemia (AML) blast cells. Ribosomes were isolated from MPC-11 cells using ultra-centrifugation. GPRE were extracted using a high KCl procedure. Blast cells from six AML patients were grown in suspension cultures for 24 and 96 h. GPRE or granulocyte macrophage-colony stimulating factor (GM-CSF) were added at the start of the incubation. GPRE, but not GM-CSF, prevented chromatin condensation and fragmentation of blast cell nuclei in AML-M2, -M4 and -M5 and the loss of nucleoli in AML-M2 and -M5. The fraction of phagocytosing blast cells in AML-M1, -M2, -M4 and -M5 was increased by GPRE. GPRE stimulated opsonin-dependent and -independent attachment and internalisation of N. meningitidis. GPRE increased the fraction of blasts expressing CD11b and CD32 in AML-M2 and -M5. GPRE diminished the fraction of AML-M5 cells bearing CD35 and CD32. GPRE also decreased the fraction of CD11c-bearing AML-M2 and -M5 cells. GM-CSF potentiated effects of GPRE in AML-M1, -M2, -M4 and -M5. GPRE and GM-CSF in combination affected phagocytosis and surface antigen expression in blast cells that were not influenced by either factor alone. Neither GPRE nor GM-CSF induced terminal differentiation or DNA-synthesis. We conclude that GPRE affects AML blast cell morphology, function and surface molecule expression, possibly by inhibiting apoptosis. The effects of GPRE may be mediated by ribosomal proteins that regulate translation and modulate the subcellular distribution of mRNA species.

The additive effect of certain drugs on the cyclosporine A inhibition of human T-cell proliferation. Studies using theophyllamine, warfarin, verapamil and dipyridamol.

The inhibitory effect of Cyclosporine A (CsA) when combined with theophyllamine, warfarin, verapamil or dipyridamol on in vitro proliferation of human T lymphocyte was investigated. All drugs caused an additional inhibition of PHA activation compared with CsA alone, also when the CD8-negative and -positive subsets were tested separately. This effect was dose-dependent for all drugs. Theophyllamine, verapamil and warfarin had to be added early during PHA-stimulated T-cell activation to cause maximal inhibition. Also when testing the proliferative response in mixed lymphocyte cultures, the drugs showed a dose-dependent additional inhibition compared with CsA alone. CsA alone caused no inhibition of lymphokine-dependent growth of T-cell lines, whereas all four drugs caused a weak inhibition in this test system.