Publications by authors named "Katharina Blatt"

50 Publications

Delineation of target expression profiles in CD34+/CD38- and CD34+/CD38+ stem and progenitor cells in AML and CML.

Blood Adv 2020 10;4(20):5118-5132

Ludwig Boltzmann Institute for Hematology and Oncology.

In an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38- and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38- and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38- LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38- cells variably expressed "aberrant" membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication-mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38- LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2020001742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594398PMC
October 2020

A hypoallergenic peptide mix containing T cell epitopes of the clinically relevant house dust mite allergens.

Allergy 2019 12 3;74(12):2461-2478. Epub 2019 Oct 3.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Background: In the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects.

Methods: IgE reactivity of HDM-allergic and non-HDM-sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty-three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively.

Results: House dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM-allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen-specific IgG levels were found in HDM allergics. PBMC from HDM-allergics produced higher levels of IL-5 whereas non-HDM-sensitized individuals mounted higher levels of IFN-gamma, IL-17, pro-inflammatory cytokines, and IL-10.

Conclusion: IgG antibodies in HDM-allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen-specific IgG antibodies do not protect against IgE-mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/all.13956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078969PMC
December 2019

Identification of a leukemia-initiating stem cell in human mast cell leukemia.

Leukemia 2019 11 5;33(11):2673-2684. Epub 2019 Apr 5.

Ludwig Boltzmann Institute for Hematology and Oncology, Medical University of Vienna, 1090, Vienna, Austria.

Mast cell leukemia (MCL) is a highly fatal malignancy characterized by devastating expansion of immature mast cells in various organs. Although considered a stem cell disease, little is known about MCL-propagating neoplastic stem cells. We here describe that leukemic stem cells (LSCs) in MCL reside within a CD34/CD38 fraction of the clone. Whereas highly purified CD34/CD38 cells engrafted NSG mice with fully manifesting MCL, no MCL was produced by CD34/CD38 progenitors or the bulk of KIT/CD34 mast cells. CD34/CD38 MCL cells invariably expressed CD13 and CD133, and often also IL-1RAP, but did not express CD25, CD26 or CLL-1. CD34/CD38 MCL cells also displayed several surface targets, including CD33, which was homogenously expressed on MCL LSCs in all cases, and the D816V mutant form of KIT. Although CD34/CD38 cells were resistant against single drugs, exposure to combinations of CD33-targeting and KIT-targeting drugs resulted in LSC-depletion and markedly reduced engraftment in NSG mice. Together, MCL LSCs are CD34/CD38 cells that express distinct profiles of markers and target antigens. Characterization of MCL LSCs should facilitate their purification and should support the development of LSC-eradicating curative treatment approaches in this fatal type of leukemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41375-019-0460-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839966PMC
November 2019

Phenotyping and Target Expression Profiling of CD34/CD38 and CD34/CD38 Stem- and Progenitor cells in Acute Lymphoblastic Leukemia.

Neoplasia 2018 06 15;20(6):632-642. Epub 2018 May 15.

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria; Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. Electronic address:

Leukemic stem cells (LSCs) are an emerging target of curative anti-leukemia therapy. In acute lymphoblastic leukemia (ALL), LSCs frequently express CD34 and often lack CD38. However, little is known about markers and targets expressed in ALL LSCs. We have examined marker- and target expression profiles in CD34/CD38 LSCs in patients with Ph ALL (n = 22) and Ph ALL (n = 27) by multi-color flow cytometry and qPCR. ALL LSCs expressed CD19 (B4), CD44 (Pgp-1), CD123 (IL-3RA), and CD184 (CXCR4) in all patients tested. Moreover, in various subgroups of patients, LSCs also displayed CD20 (MS4A1) (10/41 = 24%), CD22 (12/20 = 60%), CD33 (Siglec-3) (20/48 = 42%), CD52 (CAMPATH-1) (17/40 = 43%), IL-1RAP (13/29 = 45%), and/or CD135 (FLT3) (4/20 = 20%). CD25 (IL-2RA) and CD26 (DPPIV) were expressed on LSCs in Ph ALL exhibiting BCR/ABL1, whereas in Ph ALL with BCR/ABL1, LSCs variably expressed CD25 but did not express CD26. In Ph ALL, CD34/CD38 LSCs expressed IL-1RAP in 6/18 patients (33%), but did not express CD25 or CD26. Normal stem cells stained negative for CD25, CD26 and IL-1RAP, and expressed only low amounts of CD52. In xenotransplantation experiments, CD34/CD38 and CD34/CD38 cells engrafted NSG mice after 12-20 weeks, and targeting with antibodies against CD33 and CD52 resulted in reduced engraftment. Together, LSCs in Ph and Ph ALL display unique marker- and target expression profiles. In Ph ALL with BCR/ABL1, the LSC-phenotype closely resembles the marker-profile of CD34/CD38 LSCs in chronic myeloid leukemia, confirming the close biologic relationship of these neoplasms. Targeting of LSCs with specific antibodies or related immunotherapies may facilitate LSC eradication in ALL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neo.2018.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5994777PMC
June 2018

The KIT and PDGFRA switch-control inhibitor DCC-2618 blocks growth and survival of multiple neoplastic cell types in advanced mastocytosis.

Haematologica 2018 05 8;103(5):799-809. Epub 2018 Feb 8.

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria

Systemic mastocytosis is a complex disease defined by abnormal growth and accumulation of neoplastic mast cells in various organs. Most patients exhibit a D816V-mutated variant of , which confers resistance against imatinib. Clinical problems in systemic mastocytosis arise from mediator-related symptoms and/or organ destruction caused by malignant expansion of neoplastic mast cells and/or other myeloid cells in various organ systems. DCC-2618 is a spectrum-selective pan KIT and PDGFRA inhibitor which blocks KIT D816V and multiple other kinase targets relevant to systemic mastocytosis. We found that DCC-2618 inhibits the proliferation and survival of various human mast cell lines (HMC-1, ROSA, MCPV-1) as well as primary neoplastic mast cells obtained from patients with advanced systemic mastocytosis (IC <1 μM). Moreover, DCC-2618 decreased growth and survival of primary neoplastic eosinophils obtained from patients with systemic mastocytosis or eosinophilic leukemia, leukemic monocytes obtained from patients with chronic myelomonocytic leukemia with or without concomitant systemic mastocytosis, and blast cells obtained from patients with acute myeloid leukemia. Furthermore, DCC-2618 was found to suppress the proliferation of endothelial cells, suggesting additional drug effects on systemic mastocytosis-related angiogenesis. Finally, DCC-2618 was found to downregulate IgE-mediated histamine release from basophils and tryptase release from mast cells. Together, DCC-2618 inhibits growth, survival and activation of multiple cell types relevant to advanced systemic mastocytosis. Whether DCC-2618 is effective in patients with advanced systemic mastocytosis is currently under investigation in clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2017.179895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927976PMC
May 2018

Variability of PD-L1 expression in mastocytosis.

Blood Adv 2018 02;2(3):189-199

Department of Pathology, University of New Mexico, Albuquerque, NM.

Mastocytosis is a rare disease with heterogeneous clinical manifestations and few effective therapies. Programmed death-1 (PD-1) and its ligands (PD-L1 and PD-L2) protect tissues from immune-mediated damage and permit tumors to evade immune destruction. Therapeutic antibodies against PD-1 and PD-L1 are effective in the treatment of a variety of neoplasms. In the present study, we sought to systematically analyze expression of PD-1 and PD-L1 in a large number of patients with mastocytosis using immunohistochemistry and multiplex fluorescence staining. PD-L1 showed membrane staining of neoplastic mast cells (MCs) in 77% of systemic mastocytosis (SM) cases including 3 of 3 patients with MC leukemia, 2 of 2 with aggressive SM, 1 of 2 with smoldering SM, 3 of 4 with indolent SM, and 9 of 12 with SM with an associated hematologic neoplasm (SM component only). Ninety-two percent (23 of 25) of cutaneous mastocytosis (CM) cases and 1 of 2 with myelomastocytic leukemia expressed PD-L1, with no expression found in 15 healthy/reactive marrows, 18 myelodysplastic syndromes (MDSs), 16 myeloproliferative neoplasms (MPNs), 5 MDS/MPNs, and 3 monoclonal MC activation syndromes. Variable PD-L1 expression was observed between and within samples, with PD-L1 staining of MCs ranging from 10% to 100% (mean, 50%). PD-1 dimly stained 4 of 27 CM cases (15%), with no expression in SM or other neoplasms tested; PD-1 staining of MCs ranged from 20% to 50% (mean, 27%). These results provide support for the expression of PD-L1 in SM and CM, and PD-1 expression in CM. These data support the exploration of agents with anti-PD-L1 activity in patients with advanced mastocytosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2017011551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812326PMC
February 2018

The pan-BCL-2-blocker obatoclax (GX15-070) and the PI3-kinase/mTOR-inhibitor BEZ235 produce cooperative growth-inhibitory effects in ALL cells.

Oncotarget 2017 Sep 28;8(40):67709-67722. Epub 2017 Jun 28.

Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Vienna, Austria.

Acute lymphoblastic leukemia (ALL) is characterized by leukemic expansion of lymphoid blasts in hematopoietic tissues. Despite improved therapy only a subset of patients can be cured. Therefore, current research is focusing on new drug-targets. Members of the BCL-2 family and components of the PI3-kinase/mTOR pathway are critically involved in the regulation of growth and survival of ALL cells. We examined the effects of the pan-BCL-2 blocker obatoclax and the PI3-kinase/mTOR-inhibitor BEZ235 on growth and survival of ALL cells. In H-thymidine uptake experiments, both drugs suppressed the proliferation of leukemic cells in all patients with Philadelphia chromosome-positive (Ph) ALL and Ph ALL (obatoclax IC: 0.01-5 μM; BEZ235, IC: 0.01-1 μM). Both drugs were also found to produce growth-inhibitory effects in all Ph and all Ph cell lines tested. Moreover, obatoclax and BEZ235 induced apoptosis in ALL cells. In drug-combination experiments, obatoclax and BEZ235 exerted synergistic growth-inhibitory effects on ALL cells. Finally, we confirmed that ALL cells, including CD34/CD38 stem cells and all cell lines express transcripts for PI3-kinase, mTOR, BCL-2, MCL-1, and BCL-xL. Taken together, this data shows that combined targeting of the PI3-kinase/mTOR-pathway and BCL-2 family-members is a potent approach to counteract growth and survival of ALL cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.18810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620205PMC
September 2017

Combined targeting of STAT3 and STAT5: a novel approach to overcome drug resistance in chronic myeloid leukemia.

Haematologica 2017 09 8;102(9):1519-1529. Epub 2017 Jun 8.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Austria.

In chronic myeloid leukemia, resistance against BCR-ABL1 tyrosine kinase inhibitors can develop because of mutations, activation of additional pro-oncogenic pathways, and stem cell resistance. Drug combinations covering a broad range of targets may overcome resistance. CDDO-Me (bardoxolone methyl) is a drug that inhibits the survival of leukemic cells by targeting different pro-survival molecules, including STAT3. We found that CDDO-Me inhibits proliferation and survival of tyrosine kinase inhibitor-resistant cell lines and primary leukemic cells, including cells harboring or T315I compound mutations. Furthermore, CDDO-Me was found to block growth and survival of CD34/CD38 leukemic stem cells (LSC). Moreover, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with BCR-ABL1 tyrosine kinase inhibitors. These drug-combinations were found to block multiple signaling cascades and molecules, including STAT3 and STAT5. Furthermore, combined targeting of STAT3 and STAT5 by shRNA and STAT5-targeting drugs also resulted in synergistic growth-inhibition, pointing to a new efficient concept of combinatorial STAT3 and STAT5 inhibition. However, CDDO-Me was also found to increase the expression of heme-oxygenase-1, a heat-shock-protein that triggers drug resistance and cell survival. We therefore combined CDDO-Me with the heme-oxygenase-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects. Moreover, SMA-ZnPP was found to sensitize cells against the combination 'CDDO-Me+ tyrosine kinase inhibitor'. Together, combined targeting of STAT3, STAT5, and heme-oxygenase-1 overcomes resistance in cells, including stem cells and highly resistant sub-clones expressing BCR-ABL1 or T315I-compound mutations. Whether such drug-combinations are effective in tyrosine kinase inhibitor-resistant patients with chronic myeloid leukemia remains to be elucidated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2016.163436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685220PMC
September 2017

Expression of CD25 on leukemic stem cells in BCR-ABL1 CML: Potential diagnostic value and functional implications.

Exp Hematol 2017 07 27;51:17-24. Epub 2017 Apr 27.

Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Vienna, Austria; Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria. Electronic address:

Chronic myeloid leukemia (CML) is a stem cell-derived leukemia in which neoplastic cells exhibit the Philadelphia chromosome and the related oncoprotein BCR-ABL1. The disease is characterized by an accumulation of myeloid precursor cells in the peripheral blood and bone marrow (BM). A small fraction of neoplastic cells in the CML clone supposedly exhibits self-renewal and thus long-term disease-propagating ability. However, so far, little is known about the phenotype, function, and target expression profiles of these leukemic stem cells (LSCs). Recent data suggest that CML LSCs aberrantly express the interleukin-2 receptor alpha chain CD25. Whereas normal CD34/CD38 BM stem cells display only low amounts of CD25 or lack CD25 altogether, CD34/CD38 LSCs express CD25 strongly in more than 90% of all patients with untreated CML. As a result, CD25 can be used to identify and quantify CML LSCs. In addition, it has been shown that CD25 serves as a negative growth regulator of CML LSCs. Here, we review the value of CD25 as a novel marker and potential drug target in CML LSCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exphem.2017.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044418PMC
July 2017

Extracorporeal IgE Immunoadsorption in Allergic Asthma: Safety and Efficacy.

EBioMedicine 2017 Mar 12;17:119-133. Epub 2017 Feb 12.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria. Electronic address:

Background: Prevention of IgE-binding to cellular IgE-receptors by anti-IgE (Omalizumab) is clinically effective in allergic asthma, but limited by IgE threshold-levels. To overcome this limitation, we developed a single-use IgE immunoadsorber column (IgEnio). IgEnio is based on a recombinant, IgE-specific antibody fragment and can be used for the specific extracorporeal desorption of IgE.

Objective: To study safety and efficacy of IgEnio regarding the selective depletion of IgE in a randomized, open-label, controlled pilot trial in patients with allergic asthma and to investigate if IgEnio can bind IgE-Omalizumab immune complexes.

Methods: Fifteen subjects were enrolled and randomly assigned to the treatment group (n=10) or to the control group (n=5). Immunoadsorption was done by veno-venous approach, processing the twofold calculated plasma volume during each treatment. A minimum average IgE-depletion of 50% after the last cycle in the intention-to-treat population was defined as primary endpoint. Safety of the treatment was studied as secondary endpoint. In addition, possible changes in allergen-specific sensitivity were investigated, as well as clinical effects by peak flow measurement and symptom-recording. The depletion of IgE-Omalizumab immune complexes was studied in vitro. The study was registered at clinicaltrials.gov (NCT02096237) and conducted from December 2013 to July 2014.

Results: IgE immunoadsorption with IgEnio selectively depleted 86.2% (±5.1% SD) of IgE until the end of the last cycle (p<0.0001). Removal of pollen allergen-specific IgE was associated with a reduction of allergen-specific basophil-sensitivity and prevented increases of allergen-specific skin-sensitivity and clinical symptoms during pollen seasons. IgEnio also depleted IgE-Omalizumab immune complexes in vitro. The therapy under investigation was safe and well-tolerated. During a total of 81 aphereses, 2 severe adverse events (SAE) were recorded, one of which, an episode of acute dyspnea, possibly was related to the treatment and resolved after administration of antihistamines and corticosteroids.

Conclusions: This pilot study indicates that IgE immunoadsorption with IgEnio may be used to treat patients with pollen-induced allergic asthma. Furthermore, the treatment could render allergic patients with highly elevated IgE-levels eligible for the administration of Omalizumab and facilitate the desorption of IgE-Omalizumab complexes. This study was funded by Fresenius Medical Care Deutschland GmbH, Bad Homburg, Germany.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2017.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360571PMC
March 2017

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

EBioMedicine 2016 Sep 20;11:43-57. Epub 2016 Aug 20.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address:

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.).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2016.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5049999PMC
September 2016

Evaluation of in vitro effects of various targeted drugs on plasma cells and putative neoplastic stem cells in patients with multiple myeloma.

Oncotarget 2016 Oct;7(40):65627-65642

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria.

Multiple myeloma (MM) is a malignancy characterized by monoclonal paraproteinemia and tissue plasmocytosis. In advanced MM cytopenia and osteopathy may occur. Although several effective treatment strategies have been developed in recent years, there is still a need to identify new drug targets and to develop more effective therapies for patients with advanced MM. We examined the effects of 15 targeted drugs on growth and survival of primary MM cells and 5 MM cell lines (MM.1S, NCI-H929, OPM-2, RPMI-8226, U-266). The PI3-kinase blocker BEZ235, the pan-BCL-2 inhibitor obatoclax, the Hsp90-targeting drug 17AAG, and the Polo-like kinase-1 inhibitor BI2536, were found to exert major growth-inhibitory effects in all 5 MM cell lines tested. Moreover, these drugs suppressed the in vitro proliferation of primary bone marrow-derived MM cells and induced apoptosis at pharmacologic drug concentrations. Apoptosis-inducing effects were not only seen in the bulk of MM cells but also in MM stem cell-containing CD138-/CD20+/CD27+ memory B-cell fractions. Synergistic growth-inhibitory effects were observed in MM cell lines using various drug combinations, including 17AAG+BI2536 in MM.1S, OPM-2, RPMI-8226, and U-266 cells, 17AAG+BEZ235 in MM.1S, OPM-2, RPMI-8226, and U-266 cells, 17AAG+obatoclax in MM.1S, NCI-H929, OPM-2, and RPMI-8226 cells, BI2536+BEZ235 in MM.1S, NCI-H929, OPM-2, and RPMI-8226 cells, BI2536+obatoclax in MM.1S, OPM-2 and RPMI-8226 cells, and BEZ235+obatoclax in MM.1S and RPMI-8226 cells. Together, our data show that various targeted drugs induce profound and often synergistic anti-neoplastic effects in MM cells which may have clinical implications and may contribute to the development of novel treatment strategies in advanced MM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.11593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323180PMC
October 2016

Molecular, Structural and Immunological Characterization of Der p 18, a Chitinase-Like House Dust Mite Allergen.

PLoS One 2016 22;11(8):e0160641. Epub 2016 Aug 22.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

Background: The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated.

Objective: To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level.

Methods: Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments.

Results: Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients.

Conclusion: Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160641PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993390PMC
July 2017

Identification of CD25 as STAT5-Dependent Growth Regulator of Leukemic Stem Cells in Ph+ CML.

Clin Cancer Res 2016 Apr 25;22(8):2051-61. Epub 2015 Nov 25.

Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria. Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria.

Purpose: In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs.

Experimental Design: CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812.

Results: In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib.

Conclusions: CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-15-0767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817228PMC
April 2016

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

J Allergy Clin Immunol 2016 Feb 28;137(2):601-609.e8. Epub 2015 Oct 28.

Division of Immunopathology, Department of Pathophysiology, Center of Physiology and Pathophysiology, Vienna General Hospital (AKH), Medical University of Vienna, Vienna, Austria. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2015.08.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4748398PMC
February 2016

Identification of the Ki-1 antigen (CD30) as a novel therapeutic target in systemic mastocytosis.

Blood 2015 Dec 20;126(26):2832-41. Epub 2015 Oct 20.

Department of Internal Medicine I, Division of Hematology and Hemostaseology and Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria;

The Ki-1 antigen (CD30) is an established therapeutic target in patients with Hodgkin lymphoma and anaplastic large-cell lymphoma. We have recently shown that CD30 is expressed abundantly in the cytoplasm of neoplastic mast cells (MCs) in patients with advanced systemic mastocytosis (SM). In the current study, we asked whether CD30 is expressed on the surface of neoplastic MCs in advanced SM, and whether this surface structure may serve as therapeutic target in SM. As assessed by flow cytometry, CD30 was found to be expressed on the surface of neoplastic MCs in 3 of 25 patients (12%) with indolent SM, 4 of 7 patients (57%) with aggressive SM, and 4 of 7 patients (57%) with MC leukemia. The immature RAS-transformed human MC line MCPV-1.1 also expressed cell surface CD30, whereas the KIT-transformed MC line HMC-1.2 expressed no detectable CD30. The CD30-targeting antibody-conjugate brentuximab-vedotin inhibited proliferation in neoplastic MCs, with lower IC50 values obtained in CD30(+) MCPV-1.1 cells (10 µg/mL) compared with CD30(-) HMC-1.2 cells (>50 µg/mL). In addition, brentuximab-vedotin suppressed the engraftment of MCPV-1.1 cells in NSG mice. Moreover, brentuximab-vedotin produced apoptosis in all CD30(+) MC lines tested as well as in primary neoplastic MCs in patients with CD30(+) SM, but did not induce apoptosis in neoplastic MCs in patients with CD30(-) SM. Furthermore, brentuximab-vedotin was found to downregulate anti-IgE-induced histamine release in CD30(+) MCs. Finally, brentuximab-vedotin and the KIT D816V-targeting drug PKC412 produced synergistic growth-inhibitory effects in MCPV-1.1 cells. Together, CD30 is a promising new drug target for patients with CD30(+) advanced SM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2015-03-637728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692143PMC
December 2015

Cancer stem cells in basic science and in translational oncology: can we translate into clinical application?

J Hematol Oncol 2015 Feb 25;8:16. Epub 2015 Feb 25.

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Spitalgasse 23, Vienna, 1090, Wien, Austria.

Since their description and identification in leukemias and solid tumors, cancer stem cells (CSC) have been the subject of intensive research in translational oncology. Indeed, recent advances have led to the identification of CSC markers, CSC targets, and the preclinical and clinical evaluation of the CSC-eradicating (curative) potential of various drugs. However, although diverse CSC markers and targets have been identified, several questions remain, such as the origin and evolution of CSC, mechanisms underlying resistance of CSC against various targeted drugs, and the biochemical basis and function of stroma cell-CSC interactions in the so-called 'stem cell niche.' Additional aspects that have to be taken into account when considering CSC elimination as primary treatment-goal are the genomic plasticity and extensive subclone formation of CSC. Notably, various cell fractions with different combinations of molecular aberrations and varying proliferative potential may display CSC function in a given neoplasm, and the related molecular complexity of the genome in CSC subsets is considered to contribute essentially to disease evolution and acquired drug resistance. In the current article, we discuss new developments in the field of CSC research and whether these new concepts can be exploited in clinical practice in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13045-015-0113-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4345016PMC
February 2015

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

J Immunol 2015 Apr 18;194(8):4008-18. Epub 2015 Mar 18.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, 1090 Vienna, Austria; Christian Doppler Laboratory for Allergy Research, Medical University of Vienna, 1090 Vienna, Austria;

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1400402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415977PMC
April 2015

Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers.

J Allergy Clin Immunol 2015 Aug 11;136(2):490-3.e11. Epub 2015 Mar 11.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2015.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530582PMC
August 2015

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

J Allergy Clin Immunol 2015 May 13;135(5):1207-7.e1-11. Epub 2014 Nov 13.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2014.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418753PMC
May 2015

DPPIV (CD26) as a novel stem cell marker in Ph+ chronic myeloid leukaemia.

Eur J Clin Invest 2014 Dec;44(12):1239-45

Division of Haematology & Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria; Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria.

The concept of leukaemic stem cells (LSCs) has been developed to explain the complex cellular hierarchy and biology of leukaemias and to screen for pivotal targets that can be employed to improve drug therapies through LSC eradication in these patients. Some of the newly discovered LSC markers seem to be expressed in a disease-specific manner and may thus serve as major research tools and diagnostic parameters. A useful LSC marker in chronic myeloid leukaemia (CML) appears to be CD26, also known as dipeptidylpeptidase IV. Expression of CD26 is largely restricted to CD34(+) /CD38(-) LSCs in BCR/ABL1(+) CML, but is not found on LSCs in other myeloid or lymphoid neoplasms, with the exception of lymphoid blast crisis of CML, BCR/ABL1p210 + acute lymphoblastic leukaemia, and a very few cases of acute myeloid leukaemia. Moreover, CD26 usually is not expressed on normal bone marrow (BM) stem cells. Functionally, CD26 is a cytokine-targeting surface enzyme that may facilitate the mobilization of LSCs from the BM niche. In this article, we review our current knowledge about the biology and function of CD26 on CML LSCs and discuss the diagnostic potential of this new LSC marker in clinical haematology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/eci.12368DOI Listing
December 2014

Der p 11 is a major allergen for house dust mite-allergic patients suffering from atopic dermatitis.

J Invest Dermatol 2015 Jan 7;135(1):102-109. Epub 2014 Aug 7.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria; Christian Doppler Laboratory for the Development of Allergen Chips, Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria. Electronic address:

House dust mites (HDMs) belong to the most potent indoor allergen sources worldwide and are associated with allergic manifestations in the respiratory tract and the skin. Here we studied the importance of the high-molecular-weight group 11 allergen from Dermatophagoides pteronyssinus (Der p 11) in HDM allergy. Sequence analysis showed that Der p 11 has high homology to paramyosins from mites, ticks, and other invertebrates. A synthetic gene coding for Der p 11 was expressed in Escherichia coli and rDer p 11 purified to homogeneity as folded, alpha-helical protein as determined by circular dichroism spectroscopy. Using antibodies raised against rDer p 11 and immunogold electron microscopy, the allergen was localized in the muscle beneath the skin of mite bodies but not in feces. IgE reactivity of rDer p 11 was tested with sera from HDM-allergic patients from Europe and Africa in radioallergosorbent test-based dot-blot assays. Interestingly, we found that Der p 11 is a major allergen for patients suffering from atopic dermatitis (AD), whereas it is only a minor allergen for patients suffering from respiratory forms of HDM allergy. Thus, rDer p 11 might be a useful serological marker allergen for the identification of a subgroup of HDM-allergic patients suffering from HDM-associated AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2014.271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636057PMC
January 2015

Identification of campath-1 (CD52) as novel drug target in neoplastic stem cells in 5q-patients with MDS and AML.

Clin Cancer Res 2014 Jul 5;20(13):3589-602. Epub 2014 May 5.

Authors' Affiliations: Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Austria; Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria;

Purpose: The CD52-targeted antibody alemtuzumab induces major clinical responses in a group of patients with myelodysplastic syndromes (MDS). The mechanism underlying this drug effect remains unknown.

Experimental Design: We asked whether neoplastic stem cells (NSC) in patients with MDS (n = 29) or acute myelogenous leukemia (AML; n = 62) express CD52.

Results: As assessed by flow cytometry, CD52 was found to be expressed on NSC-enriched CD34(+)/CD38(-) cells in 8/11 patients with MDS and isolated del(5q). In most other patients with MDS, CD52 was weakly expressed or not detectable on NSC. In AML, CD34(+)/CD38(-) cells displayed CD52 in 23/62 patients, including four with complex karyotype and del(5q) and one with del(5q) and t(1;17;X). In quantitative PCR (qPCR) analyses, purified NSC obtained from del(5q) patients expressed CD52 mRNA. We were also able to show that CD52 mRNA levels correlate with EVI1 expression and that NRAS induces the expression of CD52 in AML cells. The CD52-targeting drug alemtuzumab, was found to induce complement-dependent lysis of CD34(+)/CD38(-)/CD52(+) NSC, but did not induce lysis in CD52(-) NSC. Alemtuzumab also suppressed engraftment of CD52(+) NSC in NSG mice. Finally, CD52 expression on NSC was found to correlate with a poor survival in patients with MDS and AML.

Conclusions: The cell surface target Campath-1 (CD52) is expressed on NSC in a group of patients with MDS and AML. CD52 is a novel prognostic NSC marker and a potential NSC target in a subset of patients with MDS and AML, which may have clinical implications and may explain clinical effects produced by alemtuzumab in these patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-13-2811DOI Listing
July 2014

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia.

Blood 2014 Jun 28;123(25):3951-62. Epub 2014 Apr 28.

Ludwig Boltzmann Cluster Oncology, and Department of Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Vienna, Austria;

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34(+)/CD38(─) CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1(+) cells, whereas CD26(─) SC from the same patients produced multilineage BCR/ABL1(-) engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1(+) cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2013-10-536078DOI Listing
June 2014

CD52 is a molecular target in advanced systemic mastocytosis.

FASEB J 2014 Aug 23;28(8):3540-51. Epub 2014 Apr 23.

Ludwig Boltzmann Institute of Osteology, Hanusch Hospital, Vienna, Austria

Advanced systemic mastocytosis (SM) is an aggressive hematopoietic neoplasm with poor prognosis and short survival times. So far, no curative therapy is available for affected patients. We have identified the cell surface antigen CD52 (CAMPATH-1) as a molecular target expressed abundantly on the surface of primary neoplastic mast cells (MCs) in patients with advanced SM. In contrast, neoplastic MCs of patients with indolent SM and normal MCs expressed only low levels or did not express CD52. To study the mechanisms of CD52 expression and the value of this antigen as a potential therapeutic target, we generated a human MC cell line, designated MCPV-1, by lentiviral immortalization of cord blood-derived MC progenitor cells. Functional studies revealed that activated RAS profoundly promotes surface expression of CD52. The CD52-targeting antibody alemtuzumab induced cell death in CD52(+) primary neoplastic MCs obtained from patients with SM as well as in MCPV-1 cells. NSG mice xenotransplanted with MCPV-1 cells survived significantly longer after treatment with alemtuzumab (median survival: 31 d untreated vs. 46 d treated; P=0.0012). We conclude that CD52 is a novel marker and potential therapeutic target in neoplastic MCs in patients with advanced SM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.14-250894DOI Listing
August 2014

Conversion of Der p 23, a new major house dust mite allergen, into a hypoallergenic vaccine.

J Immunol 2014 May 14;192(10):4867-75. Epub 2014 Apr 14.

Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, 1090 Vienna, Austria;

Der p 23, a new, major house dust mite (HDM) allergen that is recognized by >70% of HDM-allergic patients, has high allergenic activity and, therefore, must be considered an important component for HDM-specific immunotherapy. We constructed and characterized a hypoallergenic Der p 23 vaccine for HDM immunotherapy. Three nonallergenic peptides from the C-terminal IgE epitope-containing part of Der p 23 (P4, P5) and P6, a mutant peptide containing serines instead of cysteines, were identified. Peptides were fused to the hepatitis B virus-derived PreS domain as recombinant fusion proteins (i.e., PreS-2XP4P5 and PreS-4XP6) that were expressed in Escherichia coli and purified to homogeneity. Compared with Der p 23, PreS-2XP4P5 and PreS-4XP6 showed no relevant IgE reactivity and exhibited considerably reduced allergenic activity in basophil activation tests using blood from HDM-allergic patients. Upon immunization of rabbits, only PreS-2XP4P5 induced high levels of Der p 23-specific IgG Abs that inhibited binding of patients' IgE to Der p 23, comparable to IgG Abs induced with Der p 23, whereas Abs induced with PreS-4XP6 had only low blocking capacity. Additionally, IgG Abs induced with PreS-2XP4P5 inhibited Der p 23-induced basophil activation comparable to IgG Abs induced with Der p 23. Compared with Der p 23, PreS-2XP4P5 induced lower T cell proliferation but higher levels of the tolerogenic cytokine IL-10 and the Th1 cytokine IFN-γ in PBMCs from HDM-allergic patients, indicating an immunomodulatory capacity of the fusion protein. Therefore, PreS-2XP4P5 represents a promising candidate for immunotherapy of HDM-allergic patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1400064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582415PMC
May 2014

A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection.

Blood 2014 Jul 27;124(1):111-20. Epub 2014 Mar 27.

Laboratoire de Biologie et Pharmacologie Appliquée, Centre Nationale de la Recherche, Unité Mixte de Recherche 8113, Ecole Normale Supérieure de Cachan, Cachan, France; Laboratoire Central d'Hématologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France;

In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2013-10-534685DOI Listing
July 2014

FLAG-induced remission in a patient with acute mast cell leukemia (MCL) exhibiting t(7;10)(q22;q26) and KIT D816H.

Leuk Res Rep 2014 26;3(1):8-13. Epub 2013 Nov 26.

Institute of Pathology, Ludwig-Maximilians-University, Munich, Germany.

Mast cell leukemia (MCL) is a life-threatening disease associated with high mortality and drug-resistance. Only few patients survive more than 12 months. We report on a 55-year-old female patient with acute MCL diagnosed in May 2012. The disease was characterized by a rapid increase in white blood cells and mast cells (MC) in the peripheral blood, and a rapid increase of serum tryptase levels. The KIT D816H mutation was detected in the blood and bone marrow (BM). Induction chemotherapy with high-dose ARA-C and fludarabine (FLAG) was administered. Unexpectedly, the patient entered a hematologic remission with almost complete disappearance of neoplastic MC and a decrease of serum tryptase levels to normal range after 2 cycles of FLAG. Consecutively, the patient was prepared for allogeneic stem cell transplantation. However, shortly after the third cycle of FLAG, tryptase levels increased again, immature MC appeared in the blood, and the patient died from cerebral bleeding. Together, this case shows that intensive chemotherapy regimens, like FLAG, may induce remission in acute MCL. However, treatment responses are short-lived and the overall outcome remains dismal in these patients. We propose to separate this acute type of MCL from more subacute or chronic variants of MCL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.lrr.2013.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939382PMC
March 2014

The role of epigenetics in the regulation of apoptosis in myelodysplastic syndromes and acute myeloid leukemia.

Crit Rev Oncol Hematol 2014 Apr 12;90(1):1-16. Epub 2013 Oct 12.

Ludwig Boltzmann Cluster Oncology, Vienna, Austria; Ludwig Boltzmann Institute for Leukemia Research and Hematology, Hanusch Hospital, Vienna, Austria; 3rd Medical Department, Hanusch Hospital, Vienna, Austria.

Disordered stem cell epigenetics and apoptosis-regulating mechanisms contribute essentially to the pathogenesis of myelodysplastic syndromes (MDS) and may trigger disease-progression to secondary acute myeloid leukemia (AML). Expression of apoptosis-mediators FAS (CD95) and DAPK1 the latter being also known for its association with autophagy are upregulated in neoplastic cells in patients with low-risk MDS and epigenetically silenced and downregulated in high-risk MDS and AML as confirmed by a study 50 MDS and 30 AMLs complementing this review. 5-Azacytidine (AZA) and 5-aza-2'deoxycytidine (DAC), promoted FAS and DAPK1 gene demethylation and their (re)expression as well as apoptosis in leukemic cell lines (HL-60, KG1) which can be reversed by siRNA against FAS. Thus, promoter-demethylation of FAS and DAPK1 represents a critical mechanism of drug-induced apoptosis in neoplastic cells in MDS and AML which underscores the clinical implication of epigenetically active therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.critrevonc.2013.10.003DOI Listing
April 2014

Dissection of the IgE and T-cell recognition of the major group 5 grass pollen allergen Phl p 5.

J Allergy Clin Immunol 2014 Mar 31;133(3):836-45.e11. Epub 2013 Oct 31.

Christian Doppler Laboratory for Allergy Research, Medical University of Vienna, Vienna, Austria; Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria. Electronic address:

Background: The major timothy grass pollen allergen Phl p 5 belongs to the most potent allergens involved in hay fever and asthma.

Objective: This study characterized immune-dominant IgE- and T-cell-recognition sites of Phl p 5.

Methods: Seven peptides, P1 to P7 with a length of 31 to 38 amino acids that spanned the Phl p 5 sequence, were synthesized, characterized by circular dichroism spectroscopy, and tested for IgE reactivity, basophil activation, and T-cell reactivity. Carrier-bound peptides were studied for their ability to induce IgG antibodies in rabbits which recognize Phl p 5 or cross-reactive allergens from different grass species. Peptide-specific antibodies were tested for the capability to inhibit IgE reactivity to Phl p 5 and allergen-induced basophil activation of patients with allergy.

Results: The peptides exhibited no secondary structure and showed no IgE reactivity or relevant allergenic activity, indicating that Phl p 5 IgE epitopes are conformational. Except for P3, peptide-specific IgG antibodies blocked IgE binding to Phl p 5 of patients with allergy and cross-reacted with temperate grasses. IgE inhibition experiments and molecular modeling identified several clustered conformational IgE epitopes on the N- as well as C-terminal domain of Phl p 5. P4, which stimulated the strongest T-cell and cytokine responses in patients, was not part of the major IgE-reactive regions.

Conclusion: Our study shows an interesting dissociation of the major IgE- and T-cell-reactive domains in Phl p 5 which provides a basis for the development of novel forms of immunotherapy that selectively target IgE or T-cell responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2013.08.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624141PMC
March 2014