JAK-STAT core cancer pathway: An integrative cancer interactome analysis.

Through a comprehensive review and in silico analysis of reported data on STAT-linked diseases, we analysed the communication pathways and interactome of the seven STATs in major cancer categories and proposed rational targeting approaches for therapeutic intervention to disrupt critical pathways and addictions to hyperactive JAK/STAT in neoplastic states. Although all STATs follow a similar molecular activation pathway, STAT1, STAT2, STAT4 and STAT6 exert specific biological profiles associated with a more restricted pattern of activation by cytokines. STAT3 and STAT5A as well as STAT5B have pleiotropic roles in the body and can act as critical oncogenes that promote many processes involved in cancer development. STAT1, STAT3 and STAT5 also possess tumour suppressive action in certain mutational and cancer type context. Here, we demonstrated member-specific STAT activity in major cancer types. Through systems biology approaches, we found surprising roles for EGFR family members, sex steroid hormone receptor ESR1 interplay with oncogenic STAT function and proposed new drug targeting approaches of oncogenic STAT pathway addiction.

Metabolome and lipidome derangements during a severe mast cell activation event in a patient with indolent systemic mastocytosis.

BACKGROUND: The number of mast cells in various organs is elevated manifold in individuals with systemic mastocytosis. Degranulation can lead to life-threatening symptomatology. No data about the alterations of the metabolome and lipidome during an attack have been published. OBJECTIVE: Our aim was to analyze changes in metabolomics and lipidomics during the acute phase of a severe mast cell activation event. METHODS: A total of 43 metabolites and 11 lipid classes comprising 200 subvariants from multiple plasma samples in duplicate, covering 72 hours of a severe mast cell activation attack with nausea and vomiting, were compared with 2 baseline samples by using quantitative liquid chromatography-mass spectrometry. RESULTS: A strong enterocyte dysfunction reflected in an almost 20-fold reduction in the functional small bowel length was extrapolated from strongly reduced ornithine and citrulline concentrations and was very likely secondary to severe endothelial cell dysfunction with hypoperfusion and extensive vascular leakage. Highly increased histamine and lactate concentrations accompanied the peak in clinical symptoms. Elevated asymmetric and symmetric dimethylarginine levels combined with reduced arginine levels compromised endothelial nitric oxide synthase activity and nitric oxide signaling. Specific and extensive depletion of many lysophosphatidylcholine variants indicates localized autotaxin activation and lysophosphatidic acid release. A strong correlation of clinical parameters with histamine concentrations and symptom reduction after 100-fold elevated plasma diamine oxidase concentrations implies that histamine is the key driver of the acute phase. CONCLUSIONS: Rapid elimination of elevated histamine concentrations through use of recombinant human diamine oxidase, supplementation of lysophosphatidylcholine for immunomodulation, inhibition of autotaxin activity, and/or blockade of lysophosphatidic acid receptors might represent new treatment options for life-threatening mast cell activation events.

Preclinical human models and emerging therapeutics for advanced systemic mastocytosis.

Mastocytosis is a term used to denote a group of rare diseases characterized by an abnormal accumulation of neoplastic mast cells in various tissues and organs. In most patients with systemic mastocytosis, the neoplastic cells carry activating mutations in KIT Progress in mastocytosis research has long been hindered by the lack of suitable in vitro models, such as permanent human mast cell lines. In fact, only a few human mast cell lines are available to date: HMC-1, LAD1/2, LUVA, ROSA and MCPV-1. The HMC-1 and LAD1/2 cell lines were derived from patients with mast cell leukemia. By contrast, the more recently established LUVA, ROSA and MCPV-1 cell lines were derived from CD34+ cells of non-mastocytosis donors. While some of these cell lines (LAD1/2, LUVA, ROSAKIT WT and MCPV-1) do not harbor KIT mutations, HMC-1 and ROSAKIT D816V cells exhibit activating KIT mutations found in mastocytosis and have thus been used to study disease pathogenesis. In addition, these cell lines are increasingly employed to validate new therapeutic targets and to screen for effects of new targeted drugs. Recently, the ROSAKIT D816V subclone has been successfully used to generate a unique in vivo model of advanced mastocytosis by injection into immunocompromised mice. Such a model may allow in vivo validation of data obtained in vitro with targeted drugs directed against mastocytosis. In this review, we discuss the major characteristics of all available human mast cell lines, with particular emphasis on the use of HMC-1 and ROSAKIT D816V cells in preclinical therapeutic research in mastocytosis.

Diagnosis, management and response criteria of iron overload in myelodysplastic syndromes (MDS): updated recommendations of the Austrian MDS platform.

INTRODUCTION: Despite the availability of effective iron chelators, transfusion-related morbidity is still a challenge in chronically transfused patients with myelodysplastic syndromes (MDS). In these patients, transfusion-induced iron overload may lead to organ dysfunction or even organ failure. In addition, iron overload is associated with reduced overall survival in MDS. Areas covered: During the past 10 years, various guidelines for the management of MDS patients with iron overload have been proposed. In the present article, we provide our updated recommendations for the diagnosis, prevention and therapy of iron overload in MDS. In addition, we propose refined treatment response criteria. As in 2006 and 2007, recommendations were discussed and formulated by participants of our Austrian MDS platform in a series of meetings in 2016 and 2017. Expert commentary: Our updated recommendations should support early recognition of iron overload, optimal patient management and the measurement of clinical responses to chelation treatment in daily practice.

First-in-human response of BCL-2 inhibitor venetoclax in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive T-lymphoid malignancy usually refractory to current treatment strategies and associated with short overall survival. By applying next-generation functional testing of primary patient-derived lymphoma cells using a library of 106 US Food and Drug Administration (FDA)-approved anticancer drugs or compounds currently in clinical development, we set out to identify novel effective treatments for T-PLL patients. We found that the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax (ABT-199) demonstrated the strongest T-PLL-specific response when comparing individual ex vivo drug response in 86 patients with refractory hematologic malignancies. Mechanistically, responses to venetoclax correlated with protein expression of BCL-2 but not with expression of the BCL-2 family members myeloid cell leukemia 1 (MCL-1) and BCL-XL in lymphoma cells. BCL-2 expression was inversely correlated with the expression of MCL-1. Based on the ex vivo responses, venetoclax treatment was commenced in 2 late-stage refractory T-PLL patients resulting in clinical responses. Our findings demonstrate first evidence of single-agent activity of venetoclax both ex vivo and in humans, offering a novel agent in T-PLL.

Mechanisms, safety and efficacy of a B cell epitope-based vaccine for immunotherapy of grass pollen allergy.

BACKGROUND: We have developed a recombinant B cell epitope-based vaccine (BM32) for allergen-specific immunotherapy (AIT) of grass pollen allergy. The vaccine contains recombinant fusion proteins consisting of allergen-derived peptides and the hepatitis B surface protein domain preS as immunological carrier. METHODS: We conducted a randomized, double-blind, placebo-controlled AIT study to determine safety, clinical efficacy and immunological mechanism of three subcutaneous injections of three BM32 doses adsorbed to aluminum hydroxide versus aluminum hydroxide (placebo) applied monthly to grass pollen allergic patients (n=70). Primary efficacy endpoint was the difference in total nasal symptom score (TNSS) through grass pollen chamber exposure before treatment and 4weeks after the last injection. Secondary clinical endpoints were total ocular symptom score (TOSS) and allergen-specific skin response evaluated by titrated skin prick testing (SPT) at the same time points. Treatment-related side effects were evaluated as safety endpoints. Changes in allergen-specific antibody, cellular and cytokine responses were measured in patients before and after treatment. RESULTS: Sixty-eight patients completed the trial. TNSS significantly decreased with mean changes of -1.41 (BM32/20µg) (P=0.03) and -1.34 (BM32/40µg) (P=0.003) whereas mean changes in the BM32/10µg and placebo group were not significant. TOSS and SPT reactions showed a dose-dependent decrease. No systemic immediate type side effects were observed. Only few grade 1 systemic late phase reactions occurred in BM32 treated patients. The number of local injection site reactions was similar in actively and placebo-treated patients. BM32 induced highly significant allergen-specific IgG responses (P<0.0001) but no allergen-specific IgE. Allergen-induced basophil activation was reduced in BM32 treated patients and addition of therapy-induced IgG significantly suppressed T cell activation (P=0.0063). CONCLUSION: The B cell epitope-based recombinant grass pollen allergy vaccine BM32 is well tolerated and few doses are sufficient to suppress immediate allergic reactions as well as allergen-specific T cell responses via a selective induction of allergen-specific IgG antibodies. (ClinicalTrials.gov number, NCT01445002.).

Frequent occurrence of T cell-mediated late reactions revealed by atopy patch testing with hypoallergenic rBet v 1 fragments.

BACKGROUND: Late allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity. OBJECTIVE: We sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope-containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD). METHODS: A clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments. T-cell, cutaneous lymphocyte antigen (CLA)(+) and CCR4(+) T-cell and cytokine responses were studied by thymidine uptake, carboxyfluorescein diacetate succinimidyl ester staining, and Luminex technology, respectively. RESULTS: rBet v 1 and hypoallergenic rBet v 1 fragments induced APT reactions in not only most of the patients with birch pollen allergy with AD (11/15) but also in most of those without AD (4/5). Patients with birch pollen allergy with AD had higher Bet v 1-specific proliferation of CLA(+) and CCR4(+) T cells compared with patients with birch pollen allergy without AD. There were no differences in Bet v 1-specific CLA(+) and CCR4(+) proliferation and cytokine secretion in patients with and without APT reactions. CONCLUSION: Hypoallergenic rBet v 1 fragments induce T cell-dependent late reactions not only in patients with birch pollen allergy with AD but also in those without AD, which can be determined based on APT results but not based on in vitro parameters.

Molecular evolution of hypoallergenic hybrid proteins for vaccination against grass pollen allergy.

More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A-adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients' IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy.

Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy.

BACKGROUND: Grass pollen is one of the most important sources of respiratory allergies worldwide. OBJECTIVE: This study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach. METHODS: Fusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity. RESULTS: Ten hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation. CONCLUSION: A recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy.

Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy.

The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.

Mastocytosis: a paradigmatic example of a rare disease with complex biology and pathology.

Mastocytosis is a rare disease characterized by abnormal expansion and accumulation of tissue mast cells (MC) in one or multiple organs. In most adult patients, systemic mastocytosis (SM) is diagnosed. Based on histopathological findings and organ damage, SM is divided into indolent SM (ISM), smoldering SM (SSM), SM with an associated hematologic non-MC-lineage disease (SM-AHNMD), aggressive SM (ASM), and MC leukemia (MCL). The clinical course and prognosis vary greatly among these groups of patients. In all variants of SM and most patients, neoplastic cells display the KIT mutation D816V. This suggests that additional KIT-independent molecular defects cause progression. Indeed, additional oncogenic lesions, including RAS- and TET2 mutations, have recently been identified in advanced SM. In patients with SM-AHNMD, such additional lesions are often detectable in the 'AHNMD-component' of the disease. Clinically relevant symptoms of SM result from i) malignant MC infiltration and the subsequent organ damage seen in advanced SM and/or ii) the release of pro-inflammatory and vasoactive mediators from MC, found in all disease-variants. Therapy of SM has to be adjusted to the individual situation in each patient. In ISM, the aim is to control mediator release and mediator effects. In advanced SM, a major goal is to control MC expansion by using conventional drugs or novel targeted drugs directed against mutant forms of KIT and/or other pro-oncogenic kinase-targets. In rapidly progressing ASM, MCL and drug-resistant AHNMD, chemotherapy and subsequent stem cell transplantation has to be considered.

A nonallergenic birch pollen allergy vaccine consisting of hepatitis PreS-fused Bet v 1 peptides focuses blocking IgG toward IgE epitopes and shifts immune responses to a tolerogenic and Th1 phenotype.

Allergen-specific immunotherapy is the only allergen-specific and disease-modifying treatment for allergy. The construction and characterization of a vaccine for birch pollen allergy is reported. Two nonallergenic peptides, PA and PB, derived from the IgE-reactive areas of the major birch pollen allergen Bet v 1 were fused to the hepatitis B surface protein, PreS, in four recombinant fusion proteins containing different numbers and combinations of the peptides. Fusion proteins expressed in Escherichia coli and purified to homogeneity showed a lack of IgE reactivity and allergenic activity when tested with sera and basophils from patients allergic to birch pollen. Compared to Bet v 1 allergen, peptides PA and PB showed reduced T cell activation in PBMCs from allergic patients, whereas PreS fusion proteins induced less IL-5 and more IL-10 and IFN-γ. Immunization of rabbits with the fusion proteins, in particular with a PreS fusion protein 2PAPB-PreS, containing two copies of each peptide, induced high levels of IgG Abs against the major IgE-reactive site on Bet v 1 and related allergens. These IgG Abs inhibited allergic patients' IgE binding to Bet v 1 better than did IgG induced by immunization with complete Bet v 1. Furthermore, 2PAPB-PreS-induced IgG inhibited Bet v 1-induced basophil activation in allergic patients and CD23-facilitated allergen presentation. Our study exemplifies novel beneficial features for a PreS carrier-based peptide vaccine for birch pollen, which, in addition to the established reduction in allergenic activity, include the enhanced focusing of blocking Ab responses toward IgE epitopes, immunomodulatory activity, and reduction of CD23-facilitated allergen presentation.

Expression of a major plant allergen as membrane-anchored and secreted protein in human cells with preserved T cell and B cell epitopes.

BACKGROUND: Expression of allergens in human cells is a prerequisite for the development of antigen-specific cell therapy in IgE-mediated allergy. We developed a strategy how the clinically relevant major grass pollen allergen Phl p 5 can be efficiently secreted or expressed on the surface of human cells with preserved allergenic activity. METHODS: The cDNA of Phl p 5 was fused to a leader peptide with or without a transmembrane domain and both constructs were ligated into a mammalian expression vector. Transfection of these plasmids into human cells resulted in a membrane-anchored or secreted version of Phl p 5, respectively, as determined by ELISA or flow cytometric analysis. RESULTS: Both the secreted and membrane-anchored Phl p 5 proteins bound IgE from allergic patients in an immunoblot assay and induced specific histamine release and CD203c upregulation in basophils of grass pollen-allergic patients. Proliferation of peripheral blood mononuclear cells from Phl p 5-allergic individuals was induced upon stimulation with both variants of Phl p 5 expressed in human cells similar to recombinant Phl p 5. CONCLUSIONS: Secreted and membrane-anchored Phl p 5 expressed in human cells preserved B cell as well as T cell epitopes and may be used to develop and test various cell-based strategies for allergen-specific immunomodulation and to delineate the tolerance mechanisms involved therein.

Mapping of conformational IgE epitopes with peptide-specific monoclonal antibodies reveals simultaneous binding of different IgE antibodies to a surface patch on the major birch pollen allergen, Bet v 1.

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients' IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients' IgE binding to Bet v 1 (52-75%) were obtained with mAbs specific for two peptides comprising aa 29-58 (P2) and aa 73-103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.

Genetic engineering of trimers of hypoallergenic fragments of the major birch pollen allergen, Bet v 1, for allergy vaccination.

An immunotherapy trial performed in allergic patients with hypoallergenic recombinant fragments, comprising aa 1-74 and 75-160 of the major birch pollen allergen, Bet v 1, has indicated that the induction of allergen-specific IgG responses may be an important mechanism of this treatment. To investigate whether the immunogenicity of the rBet v 1 fragments can be increased, recombinant trimers of the fragments were produced. For this purpose, DNA trimers of rBet v 1 aa 1-74 as well as of rBet v 1 aa 75-160 were subcloned into expression plasmid pET 17b, expressed in Escherichia coli and purified. The fragments as well as the fragment trimers showed a reduced IgE-binding capacity and allergenic activity compared to rBet v 1 wildtype when tested in allergic patients. Both rBet v 1 aa 75-160 monomer and trimer induced high titers of allergen-specific IgG1 Abs in mice. Interestingly, rBet v 1 aa 1-74 trimer induced a much higher IgG(1) response to rBet v 1 than rBet v 1 aa 1-74 monomer. Consequently, IgG Abs induced with the rBet v 1 aa 1-74 trimer inhibited birch pollen allergic patients' IgE-binding 10-fold more efficiently than IgG Abs induced with the monomer. Our data show that the immunogenicity of allergy vaccines can be increased by oligomerization.

Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly.

BACKGROUND: At least 100 million patients suffer from birch pollen allergy. OBJECTIVE: Rational design of recombinant derivatives of the major birch pollen allergen, Bet v 1, characterized by reduced IgE reactivity, preservation of sequences relevant for the induction of allergen-specific blocking IgG, and maintenance of T-cell epitopes for immunotherapy of birch pollen allergy. METHODS: Three recombinant mosaic proteins derived from Bet v 1 were generated by reassembly of codon-optimized genes coding for Bet v 1 fragments containing the elements for the induction of allergen-specific blocking IgG antibodies and the major T-cell epitopes. The proteins were expressed in Escherichia coli as recombinant mosaic molecules and compared with the Bet v 1 wild-type protein by chemical and structural methods, regarding IgE-binding and IgG-binding capacity, in basophil activation assays and tested for the in vivo induction of IgG responses. RESULTS: Three recombinant Bet v 1 (rBet v 1) mosaic proteins with strongly reduced IgE reactivity and allergenic activity were expressed and purified. Immunization with the recombinant hypoallergens induced IgG antibodies that inhibited IgE reactivity of patients with allergy to Bet v 1 comparable to those induced with the rBet v 1 wild-type allergen. CONCLUSION: We report the generation and preclinical characterization of 3 hypoallergenic rBet v 1 derivatives with suitable properties for immunotherapy of birch pollen allergy.

Polo-like kinase 1 (Plk1) as a novel drug target in chronic myeloid leukemia: overriding imatinib resistance with the Plk1 inhibitor BI 2536.

In most patients with chronic myeloid leukemia (CML), the disease can be kept under control using the BCR/ABL kinase inhibitor imatinib. Nevertheless, resistance or intolerance to imatinib and other BCR/ABL inhibitors may occur during therapy. Therefore, CML research is focusing on novel targets and targeted drugs. Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an essential role in mitosis. In this study, we examined the expression of Plk1 in CML cells and its potential role as a therapeutic target. Plk1 was found to be expressed in phosphorylated form in the CML cell line K562 as well as in primary CML cells in all patients tested. Inhibition of BCR/ABL by imatinib or nilotinib (AMN107) led to decreased expression of the Plk1 protein in CML cells, suggesting that BCR/ABL promotes Plk1 generation. Silencing of Plk1 in CML cells by a small interfering RNA approach was followed by cell cycle arrest and apoptosis. Furthermore, the Plk1-targeting drug BI 2536 was found to inhibit proliferation of imatinib-sensitive and imatinib-resistant CML cells, including leukemic cells, carrying the T315 mutation of BCR/ABL with reasonable IC(50) values (1-50 nmol/L). The growth-inhibitory effects of BI 2536 on CML cells were found to be associated with cell cycle arrest and apoptosis. Moreover, BI 2536 was found to synergize with imatinib and nilotinib in producing growth inhibition in CML cells. In conclusion, Plk1 is expressed in CML cells and may represent a novel, interesting target in imatinib-sensitive and imatinib-resistant CML.

Red blood cell transfusion dependence and outcome after allogeneic peripheral blood stem cell transplantation in patients with de novo myelodysplastic syndrome (MDS).

The prognosis of patients with de novo myelodysplastic syndrome (MDS) who are red blood cell transfusion-dependent (TD) and receive supportive care is inferior to that of patients who do not require transfusions. Whether TD also affects outcome after allogeneic transplantation is unknown. Consequently, in 172 de novo MDS patients (median age, 51 years), we analyzed the impact of TD on outcome after high-dose conditioning and allogeneic peripheral blood stem cell transplantation (PBSCT). With a median follow-up of 37 months, the probability of 3-year overall survival (OS) did not differ significantly between patients who were TD and those who were not TD before PBSCT (P=.1); however, transfusion burden, as reflected by ferritin levels, was correlated with a greater probability of severe acute graft-versus-host disease (aGVHD; P=.03) and a higher comorbidity index (P=.01), and OS was inferior in those patients with a ferritin level>1000 microg/L before PBSCT (P=.03). In multivariate analysis, only marrow myeloblast count (P=.01) and comorbidity index (P=.001) had a significant impact on OS. Our data do not identify TD as an independent negative prognostic factor for outcome after allogeneic PBSCT' however, iron overload (presumably transfusion-related) may contribute to poor transplantation success by adding to the overall comorbidities. Whether clinical intervention in the form of iron chelation can improve the outcome of allogeneic PBSCT in TD patients with MDS remains to be determined.

Disruption of allergenic activity of the major grass pollen allergen Phl p 2 by reassembly as a mosaic protein.

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.

A hypoallergenic hybrid molecule with increased immunogenicity consisting of derivatives of the major grass pollen allergens, Phl p 2 and Phl p 6.

Allergen-specific immunotherapy is currently based on the administration of allergen extracts containing natural allergens. However, its broad application is limited by the poor quality of these extracts. Based on recombinant allergens, well-defined allergy vaccines for allergen-specific immunotherapy can be produced. Furthermore, they can be modified to reduce their allergenic activity and to avoid IgE-mediated side effects. Here, we demonstrate that the immunogenicity of two grass pollen-derived hypoallergenic allergen derivatives could be increased by engineering them as a single hybrid molecule. We used a hypoallergenic Phl p 2 mosaic, generated by fragmentation of the Phl p 2 sequence and reassembly of the resulting peptides in an altered order, and a truncated Phl p 6 allergen, to produce a hybrid protein. The hybrid retained the reduction of IgE reactivity and allergenic activity of its components as shown by ELISA and basophil activation assays. Immunization with the hybrid molecule demonstrated the increased immunogenicity of this molecule, leading to higher levels of allergen-specific IgG antibodies compared to the single components. These antibodies could inhibit patients' IgE binding to the wild-type allergens. Thus, the described strategy allows the development of safer and more efficacious vaccines for the treatment of grass pollen allergy.