Targeting of mTOR is associated with decreased growth and decreased VEGF expression in acute myeloid leukaemia cells.

BACKGROUND: The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour-associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF-expression in acute myeloid leukaemia (AML). Three mTOR-targeting drugs, rapamycin, everolimus (RAD001) and CCI-779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined. MATERIALS AND METHODS: Growth of AML cells and AML-derived cell lines was assessed by (3)H-thymidine incorporation, survival was examined by light- and electron microscopy, by Tunel assay and by AnnexinV-staining, and the expression of VEGF by Northern blotting, RT-PCR and ELISA. RESULTS: Rapamycin was found to counteract growth in the AML cell lines U937 and KG1a as well as in primary AML cells in 14/18 patients examined. The effects of rapamycin and its derivatives were dose-dependent (IC(50): 10 pM-100 nM). It was also found that exposure to mTOR-targeting drugs resulted in apoptosis and in decreased expression of VEGF in leukaemic cells. CONCLUSIONS: mTOR-targeting drugs exert antileukaemic effects on AML cells in vitro through multiple actions, including direct inhibition of proliferation, induction of apoptosis and suppression of VEGF. Based on this study and other studies, mTOR can be regarded as a potential drug target in AML.

Reducing allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccination.

BACKGROUND: The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy. METHODS: A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice. RESULTS: Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients' IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils. CONCLUSION: We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.

Mixed-lineage eosinophil/basophil crisis in MDS: a rare form of progression.

BACKGROUND: Basophilic crisis and eosinophilia are well recognized features of advanced chronic myeloid leukaemia. In other myeloid neoplasms, however, transformation with marked basophilia and eosinophilia is considered unusual. DESIGN: We examined the long-term follow-up of 322 patients with de novo myelodysplastic syndromes (MDS) to define the frequency of basophilic, eosinophilic and mixed lineage (basophilic and eosinophilic) transformation. RESULTS: Of all patients, only one developed mixed lineage crisis (>or= 20% basophils and >or= 20% eosinophils). In this patient, who initially suffered from chronic myelomonocytic leukaemia, basophils increased to 48% and eosinophils up to 31% at the time of progression. Mixed lineage crisis was not accompanied by an increase in blast cells or organomegaly. The presence of BCR/ABL and other relevant fusion gene products (FIP1L1/PDGFRA, AML1/ETO, PML/RAR alpha, CBF beta/MYH11) were excluded by PCR. Myelomastocytic transformation/myelomastocytic leukaemia and primary mast cell disease were excluded by histology, KIT mutation analysis, electron microscopy and immunophenotyping. Basophils were thus found to be CD123+, CD203c+, BB1+, KIT- cells, and to express a functional IgE-receptor. Among the other patients with MDS examined, 4(1.2%) were found to have marked basophilia (>or= 20%) and 7(2.1%) were found to have massive eosinophilia ( >or= 20%), whereas mixed-lineage crisis was detected in none of them. CONCLUSIONS: Mixed basophil/eosinophil crisis may develop in patients with MDS but is an extremely rare event.

Recombinant allergens promote expression of aminopeptidase-n (CD13) on basophils in allergic patients.

IgE-dependent activation of basophils is associated with upregulation of several surface molecules. We recently identified the surface enzyme aminopeptidase N (CD13) as a novel activation antigen on human basophils. In the present study, we asked whether CD13 can be employed as a novel marker of allergen-induced activation of basophils in allergic individuals. Patients allergic to major allergens from grass pollen (Phl p 1, Phl p 5), birch pollen (Bet v 1), or house dust mites (Der p 2), were examined. Blood basophils were exposed to various concentrations of recombinant allergens for 15 minutes, and examined for expression of CD13 by multicolor flow cytometry. The allergen-induced upregulation of CD13 was compared with allergen-dependent increases in expression of CD63 and CD203c. Exposure to recombinant allergens resulted in an increase in expression of CD13 on basophils in all sensitized individuals, whereas no increase in CD13 was seen in healthy controls. The effects of the recombinant allergens on CD13-expression were dose- and time-dependent, were not observed in the absence of extracellular calcium, and were counteracted by preincubation of basophils with the PI3-kinase-targeting drugs staurosporin and LY294002. There was a good correlation between allergen-induced upregulation of CD13, CD63, and CD203c on basophils. In aggregate, our data show that recombinant allergens promote expression of CD13 on basophils in sensitized individuals. The functional significance and diagnostic implications of this observation remain to be determined.

Immunohistochemical assessment of CD25 is equally sensitive and diagnostic in mastocytosis compared to flow cytometry.

BACKGROUND: Systemic mastocytosis (SM) is a clonal myeloid disorder characterized by abnormal accumulation and growth of mast cells (MC) in internal organs. In most cases, the bone marrow is involved. Expression of CD25 in bone marrow MC, with or without coexpression of CD2, is an important minor SM criterion. So far, most studies have examined CD25-expression on MC by flow cytometry. MATERIALS AND METHODS: We examined the expression of CD25 in MC in patients with SM (n = 25) by immunohistochemistry (IHC) and compared these data with results obtained by flow cytometric assessment of CD25-expression. In addition, we compared CD25-staining results with that obtained with an antibody against CD2. RESULTS: In a majority of all patients (> 80%), CD25 was detectable by both staining techniques. However, in one patient, CD25 was only detectable on MC by IHC, but not by flow cytometry, and in two patients in whom IHC could not be applied because of lack of compact MC infiltrates, flow cytometry revealed aberrant expression of CD25. The antibody against CD2 produced diagnostic staining results in a smaller group of patients (flow cytometry: 65%; IHC: 28% of SM cases) compared to CD25 (> 80%). CONCLUSIONS: CD25-IHC is equally diagnostic and sensitive in SM compared to flow cytometry and thus can be recommended as a diagnostic test. Our data also suggest that the diagnostic value of CD25 exceeds that of CD2, and that optimal assessment of CD25-expression in neoplastic MC in all patients requires the application of both techniques, flow cytometry and IHC.

CD203c is overexpressed on neoplastic mast cells in systemic mastocytosis and is upregulated upon IgE receptor cross-linking.

The ectoenzyme E-NPP3 (CD203c) has recently been identified as a novel activation-linked cell surface antigen on basophils. In the present study, we examined expression of CD203c on normal mast cells (MC)and bone marrow (bm) MC derived from 85 patients with systemic mastocytosis (SM), including cases with indolent SM (ISM, n=72), SM with associated clonal hematologic non-MC-lineage disease (SM-AHNMD, n=6), aggressive SM (ASM, n=3), and mast cell leukemia (MCL, n=4). Surface expression of CD203c was analyzed by multicolor flow cytometry. In patients with SM, bm MC expressed significantly higher amounts of CD203c compared to normal bm MC (median MFI in controls: 260 versus median MFI in SM: 516, p<0.05). Slightly lower amounts of CD203c were detected on MC in SM-AHNMD and ASM compared to ISM. To demonstrate CD203c expression in MC at the mRNA level, neoplastic MC were highly enriched by cell sorting, and were found to express CD203c mRNA in RT-PCR analysis. Cross-linking of the IgE receptor on MC resulted in a substantial upregulation of CD203c, whereas the KIT-ligand stem cell factor (SCF) showed no significant effects. In conclusion, CD203c is a novel activation-linked surface antigen on MC that is upregulated in response to IgE receptor cross-linking and is overexpressed on neoplastic MC in patients with SM.

Interleukin-3 promotes the expression of E-NPP3/CD203C on human blood basophils in healthy subjects and in patients with birch pollen allergy.

We recently identified the ectoenzyme CD203c as a novel basophil activation antigen that is upregulated in response to FcepsilonRI cross-linkage. We investigated the effects of various interleukins (ILs) on expression of CD203c on blood basophils using an antibody against CD203c and flow cytometry. Of all cytokines tested, only IL-3 was found to upregulate expression of CD203c on basophils above baseline levels. The effects of IL-3 were dose- and time-dependent (EC(50): 0.1-1 ng/ml) without differences observed between healthy and allergic donors. Whereas anti-IgE induced maximum upregulation of CD203c within 15 minutes, the IL-3-induced upregulation showed a maximum after 180 minutes. IgE-receptor cross-linking resulted in enhanced expression of both CD63 and CD203c, whereas IL-3 enhanced the levels of CD203c without promoting expression of CD63. The IL-3-induced upregulation of CD203c was also observed in highly enriched basophils and was counteracted by a blocking antibody against the alpha chain of the IL-3 receptor (CD123). The IL-3-induced upregulation of CD203c was also found to depend on the presence of calcium. To analyze signaling pathways involved in IL-3-induced upregulation of CD203c, pharmacologic inhibitors were applied. The PI3-kinase inhibitors, wortmannin and LY294002 counteracted the IL-3-induced expression of CD203c, whereas MEK- and PKC inhibitors showed no effects. In conclusion, IL-3 upregulates expression of CD203c on basophils through a specific receptor and via a PI3-kinase-dependent signaling-pathway. Compared to FcepsilonRI-mediated cell activation, IL-3-induced upregulation of CD203c is a late(r) event and is not accompanied by upregulation of CD63.

Detection of novel leukocyte differentiation antigens on basophils and mast cells by HLDA8 antibodies.

BACKGROUND: Basophils (BA) and mast cells (MC) are important effector cells in allergic reactions. Development, growth and effector cell functions are regulated by a network of cytokines, other ligands, and respective cell surface antigens. METHODS: We examined the expression of novel CD antigens on human BA, lung MC, the BA cell line KU-812, and the MC line HMC-1. Expression of surface antigens was analyzed by monoclonal antibodies (mAb) of the HLDA8 workshop and flow cytometry. RESULTS: Basophils were found to stain positive for CXCR1 (CD181), CCR1 (CD191), CCR2 (CD192), CCR7 (CD197), IL-18Ralpha (CDw218a), IL-18Rbeta (CDw218b), TRAIL-R1 (CD261), TRAIL-R2 (CD262), TACI (CD267), TLR-4 (CD284), LAIR1 (CD305), EMR-2 (CD312), JAM1 (CD321), and JAM2 (CD322). Lung MC were found to react with mAb against EMR-2 (CD312) and JAM1 (CD321). KU-812 cells were found to stain positive for CXCR1 (CD181), TRAIL-R2 (CD262), B7H2 (CD275), TLR-4 (CD284), JAM1 (CD321), and E-Cadherin (CD324). HMC-1 cells were recognized by mAb against TRAIL-R2 (CD262), B7H2 (CD275), LAIR1 (CD305), EMR-2 (CD312), JAM1 (CD321), and Siglec-6 (CDw327). CONCLUSIONS: Extensive phenotyping with antibodies against novel CD antigens provides further evidence that BA and MC represent two separate hematopoietic cell lineages with unique phenotypic properties observed in mature cells as well as malignant immature cells. Further studies are required to define the functional role of these CD antigens expressed in BA and MC.

Effects of various statins on cytokine-dependent growth and IgE-dependent release of histamine in human mast cells.

BACKGROUND: Statins are inhibitors of hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, a key enzyme in mevalonic acid (MVA)-dependent signaling. Recent data suggest that statins exhibit profound inhibitory effects on growth and function of various immune cells. In the present study, we examined the in vitro effects of five different statins on primary human mast cells (MCs), MC progenitors, and the human MC line HMC-1. METHODS: Histamine release experiments were conducted on isolated MCs using statins and an anti-immunoglobulin E (IgE) antibody. Culture experiments were performed with stem cell factor (SCF) and interleukin (IL)-6, and cord blood-derived progenitors. RESULTS: Preincubation of primary lung MCs with cerivastatin or atorvastatin (1-50 microM) for 24 h resulted in inhibition of anti-IgE-induced release of histamine. The effects of both statins were dose-dependent. Moreover, both statins, and to a lesser degree lovastatin, were found to inhibit the SCF-induced differentiation of MCs from their progenitors. The other statins tested (simvastatin, pravastatin) did not affect mediator release or growth of MCs. CONCLUSIONS: Cerivastatin and atorvastatin act as inhibitors of growth and function of human MCs.