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Background. Clinical and epidemiological studies have suggested the development of acquired immunity in individuals previously infected with Giardia lamblia. However, there are no data on the long-term cellular immunity and genotype cross-reactivity. An outbreak of assemblage B giardiasis in a nonendemic area made it possible to evaluate the long-term cellular mediated immunity and its specificity toward the 2 Giardia assemblages known to infect humans. Methods. Peripheral blood mononuclear cells from 19 individuals infected with Giardia assemblage B 5 years previously and from 10 uninfected controls were cultured with antigens from assemblage A and B Giardia trophozoites for 6 days. Cell-mediated immunity was measured by a (3)H-thymidine proliferation assay and flow cytometric analysis of activation markers HLA-DR, CD45RO, CD25, and CD26 in T-cell subsets. Results. Proliferation responses were significantly elevated in the group previously exposed to Giardia for nearly all Giardia antigens tested. Individual responses toward Giardia trophozoite whole cell, cytosolic, and excretory-secretory antigens from both assemblages correlated well. Activation marker responses were mainly seen in CD4 T cells. Conclusions. G. lamblia infection induces long-term, albeit variable, cellular immune responses that are not assemblage specific and that are largely driven by CD4 T-cell activation. [ABSTRACT FROM AUTHOR]

Examines the effects of a fatty acid analogue, tetradecylthioacetic acid (TTA), on the functional phenotype of native, human acute myeloid leukemia (AML) cells. Healthy individuals receiving oral TTA treatment; Preparation and culture of normal peripheral blood mononuclear cells; Preparation of AML blasts.

The systemic levels of many soluble adhesion molecules and cytokines are altered in hematologic malignancies, autoimmune diseases and serious infections. Among mediators that show altered serum levels are soluble (S) L-selectin (CD63L) and soluble intercellular adhesion molecule-1 (ICAM-1/CD54) expressed by immunocompetent and hematopoietic cells and the hematopoietic growth factor thrombopoietin (Tpo). In the present study, we investigated whether the serum levels of these mediators show circadian variations in healthy individuals. Venous blood serum levels of all three mediators showed only minimal variations when measured regularly during a 24-h period. In contrast, previous studies have demonstrated that the systemic levels of these mediators are markedly altered during serious bacterial infection (SL-selectin, sICAM-1) and severe thrombocytopenia (Tpo). The results suggest that serum sampling at standardized times is probably not required in the future clinical evaluation of these three parameters. [ABSTRACT FROM AUTHOR]

Focuses on a study which examined the robustness of human CD34 precursor cells concerning the freeze/thaw procedure. Materials and methods; Results; Discussion.

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. [ABSTRACT FROM AUTHOR]

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 chemotherapyinduced 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. [ABSTRACT FROM AUTHOR]

Examines the risk factors and complicating infections in leukemia patients with chemotherapy induced neutropenia. Description of leukemia patients with neutropenia; Treatment of leukemia.

Analyzes serum thrombopoietin levels in acute leukemia patients with therapy-induced cytopenia. Inverse correlation between the serum levels and platelets counts in thrombocytopenic patients; Increase in the serum levels with development of complicating bacterial infections in the patients; Impact of the acute phase reaction on the serum levels.

The initial evaluation of patients with suspected deep vein thrombosis includes the use of biomarkers reflecting activation of the coagulation system. However, the thromboembolic process and neighboring inflammatory responses also affect endothelial cells, and endothelial cell markers may therefore be altered by the disease. In the present population-based single-center study, we investigated the plasma levels of the endothelium-specific biomarkers soluble E-selectin and endocan in a consecutive and unselected group of 120 patients admitted to hospital for suspected deep vein thrombosis. Blood samples were collected when patients arrived at the hospital. DVT patients showed evidence for an acute phase reaction with increased serum C-reactive protein levels, but this was similar to many other patients admitted with suspected but not verified thrombosis. Plasma endocan and E-selectin levels did not differ between patients with thrombosis, healthy controls and the patients without verified thrombosis (i.e. patients with other causes of their symptoms, including various inflammatory and non-inflammatory conditions). However, the combined use of endothelial biomarkers, C-reactive protein and D-dimer could be used to identify patient subsets with different frequencies of venous thrombosis. Thus, analysis of plasma biomarker profiles including endothelial cell markers may be helpful in the initial evaluation of patients with deep vein thrombosis.

As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super-SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm-13, NB4, MV4-11, THP-1, and OCI-AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy., Results: Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells., Conclusions: Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.

The combined use of the histone deacetylase inhibitor valproic acid (VPA), the retinoic acid receptor- α agonist all-trans retinoic acid (ATRA), and the deoxyribonucleic acid polymerase- α inhibitor cytarabine (Ara-C) is now considered for disease-stabilizing treatment of acute myeloid leukemia (AML). Leukemogenesis and leukemia cell chemoresistance seem to be supported by neighbouring stromal cells in the bone marrow, and we have therefore investigated the effects of these drugs on primary human endothelial cells and the osteoblastic Cal72 cell line. The results show that VPA and Ara-C have antiproliferative effects, and the antiproliferative/cytotoxic effect of Ara-C was seen at low concentrations corresponding to serum levels found during low-dose in vivo treatment. Furthermore, in functional assays of endothelial migration and tube formation VPA elicited an antiangiogenic effect, whereas ATRA elicited a proangiogenic effect. Finally, VPA and ATRA altered the endothelial cell release of angiogenic mediators; ATRA increased levels of CXCL8, PDGF-AA, and VEGF-D, while VPA decreased VEGF-D and PDGF-AA/BB levels and both drugs reduced MMP-2 levels. Several of these mediators can enhance AML cell proliferation and/or are involved in AML-induced bone marrow angiogenesis, and direct pharmacological effects on stromal cells may thus indirectly contribute to the overall antileukemic activity of this triple drug combination.

Endothelial cells are involved in the pathogenesis of acute graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. These cells express several molecules that can be detected as biologically active soluble forms; serum levels of these molecules may thereby reflect the functional status of endothelial cells. Furthermore, acute GVHD is an inflammatory reaction and endothelial cells function as local regulators of inflammation. We therefore investigated whether differences in preconditioning/pretransplant serum levels of endothelium-expressed molecules (i.e., endocan, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin) were associated with a risk of posttransplant GVHD. Our study should be regarded as a population-based study of consecutive and thereby unselected patients (n = 56). Analysis of this pretreatment endothelium biomarker profile by unsupervised hierarchical clustering identified a subset of patients with increased early nonrelapse mortality. Furthermore, low endocan levels were significantly associated with acute GVHD in the liver and gastrointestinal tract, whereas high VCAM-1 levels were associated with acute GVHD in the skin only. Our study suggests that the preconditioning/pretransplant status of endothelial cells (possibly through altered trafficking of immunocompetent cells) is important for the risk and the organ involvement of later acute GVHD.

Background: Arginine is a conditionally essential amino acid important in growing individuals and under non-homeostatic conditions/disease. Many pathogens interfere with arginine-utilization in host cells, especially nitric oxide (NO) production, by changing the expression of host enzymes involved in arginine metabolism. Here we used human intestinal epithelial cells (IEC) and three different isolates of the protozoan parasite Giardia intestinalis to investigate the role of arginine and arginine-metabolizing enzymes during intestinal protozoan infections., Results: RNA expression analyses of major arginine-metabolizing enzymes revealed the arginine-utilizing pathways in human IECs (differentiated Caco-2 cells) grown in vitro. Most genes were constant or down-regulated (e.g. arginase 1 and 2) upon interaction with Giardia, whereas inducible NO synthase (iNOS) and ornithine decarboxylase (ODC) were up-regulated within 6 h of infection. Giardia was shown to suppress cytokine-induced iNOS expression, thus the parasite has both iNOS inducing and suppressive activities. Giardial arginine consumption suppresses NO production and the NO-degrading parasite protein flavohemoglobin is up-regulated in response to host NO. In addition, the secreted, arginine-consuming giardial enzyme arginine deiminase (GiADI) actively reduces T-cell proliferation in vitro. Interestingly, the effects on NO production and T cell proliferation could be reversed by addition of external arginine or citrulline., Conclusions: Giardia affects the host's arginine metabolism on many different levels. Many of the effects can be reversed by addition of arginine or citrulline, which could be a beneficial supplement in oral rehydration therapy.

Background: A large proportion of patients with acute myeloid leukemia (AML) are not fit for intensive and potentially curative therapy due to advanced age or comorbidity. Previous studies have demonstrated that a subset of these patients can benefit from disease-stabilizing therapy based on all-trans retinoic acid (ATRA) and valproic acid. Even though complete hematological remission is only achieved for exceptional patients, a relatively large subset of patients respond to this treatment with stabilization of normal peripheral blood cell counts., Methods: In this clinical study we investigated the efficiency and safety of combining (i) continuous administration of valproic acid with (ii) intermittent oral ATRA treatment (21.5 mg/m2 twice daily) for 14 days and low-dose cytarabine (10 mg/m2 daily) for 10 days administered subcutaneously. If cytarabine could not control hyperleukocytosis it was replaced by hydroxyurea or 6-mercaptopurin to keep the peripheral blood blast count below 50 × 109/L., Results: The study included 36 AML patients (median age 77 years, range 48 to 90 years) unfit for conventional intensive chemotherapy; 11 patients responded to the treatment according to the myelodysplastic syndrome (MDS) response criteria and two of these responders achieved complete hematological remission. The most common response to treatment was increased and stabilized platelet counts. The responder patients had a median survival of 171 days (range 102 to > 574 days) and they could spend most of this time outside hospital, whereas the nonresponders had a median survival of 33 days (range 8 to 149 days). The valproic acid serum levels did not differ between responder and nonresponder patients and the treatment was associated with a decrease in the level of circulating regulatory T cells., Conclusion: Treatment with continuous valproic acid and intermittent ATRA plus low-dose cytarabine has a low frequency of side effects and complete hematological remission is seen for a small minority of patients. However, disease stabilization is seen for a subset of AML patients unfit for conventional intensive chemotherapy.