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Objective: This study aims to explore the potential of organic electrolytic photocapacitors (OEPCs), an innovative photovoltaic device, in mediating the activation of native voltage-gated Cav1.2 channels (ICa,L) in Guinea pig ventricular cardiomyocytes. Methods: Whole-cell patch-clamp recordings were employed to examine light-triggered OEPC mediated ICa,L activation, integrating the channel's kinetic properties into a multicompartment cell model to take intracellular ion concentrations into account. A multidomain model was additionally incorporated to evaluate effects of OEPC-mediated stimulation. The final model combines external stimulation, multicompartmental cell simulation, and a patch-clamp amplifier equivalent circuit to assess the impact on achievable intracellular voltage changes. Results: Light pulses activated ICa,L, with amplitudes similar to voltage-clamp activation and high sensitivity to the L-type Ca2+ channel blocker, nifedipine. Light-triggered ICa,L inactivation exhibited kinetic parameters comparable to voltage-induced inactivation. Conclusion: OEPC-mediated activation of ICa,L demonstrates their potential for nongenetic optical modulation of cellular physiology potentially paving the way for the development of innovative therapies in cardiovascular health. The integrated model proves the light-mediated activation of ICa,L and advances the understanding of the interplay between the patch-clamp amplifier and external stimulation devices. Significance: Treating cardiac conduction disorders by minimal-invasive means without genetic modifications could advance therapeutic approaches increasing patients’ quality of life compared with conventional methods employing electronic devices.

To study the longitudinal performance of fully automated cartilage segmentation in knees with radiographic osteoarthritis (OA), we evaluated the sensitivity to change in progressor knees from the Foundation for the National Institutes of Health OA Biomarkers Consortium between the automated and previously reported manual expert segmentation, and we determined whether differences in progression rates between predefined cohorts can be detected by the fully automated approach. The OA Initiative Biomarker Consortium was a nested case–control study. Progressor knees had both medial tibiofemoral radiographic joint space width loss (≥0.7 mm) and a persistent increase in Western Ontario and McMaster Universities Osteoarthritis Index pain scores (≥9 on a 0–100 scale) after 2 years from baseline (n = 194), whereas non‐progressor knees did not have either of both (n = 200). Deep‐learning automated algorithms trained on radiographic OA knees or knees of a healthy reference cohort (HRC) were used to automatically segment medial femorotibial compartment (MFTC) and lateral femorotibial cartilage on baseline and 2‐year follow‐up magnetic resonance imaging. Findings were compared with previously published manual expert segmentation. The mean ± SD MFTC cartilage loss in the progressor cohort was –181 ± 245 μm by manual segmentation (standardized response mean [SRM] –0.74), –144 ± 200 μm by the radiographic OA–based model (SRM –0.72), and –69 ± 231 μm by HRC‐based model segmentation (SRM –0.30). Cohen's dfor rates of progression between progressor versus the non‐progressor cohort was –0.84 (P< 0.001) for manual, –0.68 (P< 0.001) for the automated radiographic OA model, and –0.14 (P= 0.18) for automated HRC model segmentation. A fully automated deep‐learning segmentation approach not only displays similar sensitivity to change of longitudinal cartilage thickness loss in knee OA as did manual expert segmentation but also effectively differentiates longitudinal rates of loss of cartilage thickness between cohorts with different progression profiles.

This paper presents applications of control system theory in biomedical engineering. These methodologies are used in engineering sciences to obtain a mathematical model of systems, but system identification as scientific methodology is rarely used in biomedical engineering. The paper presents exemplarily control theory and system identification as methods for obtaining a mathematical model of the spread SARS-CoV-2 virus. The models obtained in the course of this are data-driven and strongly data-dependent. The available dataset allowed us to consider a model of a pandemic spread in the context of both the number of tested individuals and the number of infected individuals and with a resultant model that is nonlinear. We also considered a mathematical model for the dependence between the number of confirmed infected individuals and the number of deaths caused by the disease. The resulting model is linear given with the transfer function corresponding to the second-order differential equation. The mathematical models developed were additionally analyzed in accordance with controllability and observability.

Transport is non-reciprocal when not only the sign, but also the absolute value of the current depends on the polarity of the applied voltage. It requires simultaneously broken inversion and time-reversal symmetries, for example, by an interplay of spin–orbit coupling and magnetic field. Hitherto, observation of nonreciprocity was tied to resistivity, and dissipationless non-reciprocal circuit elements were elusive. Here we engineer fully superconducting non-reciprocal devices based on highly transparent Josephson junctions fabricated on InAs quantum wells. We demonstrate supercurrent rectification far below the transition temperature. By measuring Josephson inductance, we can link the non-reciprocal supercurrent to an asymmetry of the current–phase relation, and directly derive the supercurrent magnetochiral anisotropy coefficient. A semiquantitative model explains well the main features of our experimental data. Non-reciprocal Josephson junctions have the potential to become for superconducting circuits what pn junctions are for traditional electronics, enabling new non-dissipative circuit elements.

AbstractAims:Modern pharmaceutical product development is a long and complex process associated with significant investments by pharmaceutical companies. The innovative pharmaceutical industry accounts for the vast majority of expenditures in clinical trials of potential new pharmaceuticals and therefore generates economic activity within a country. The aim was to assess the far-reaching economic impact of industry-sponsored clinical-trials (ISCTs) of pharmaceutical products for the healthcare system and the national economy.Materials and methods:The study approach was based on three analytical steps. First, a survey among 15 pharmaceutical companies in Austria was conducted to evaluate the annual number of ISCTs subdivided according to trial phase, therapeutic areas and associated employees. Second, the monetary value of treatments performed in ISCTs was calculated based on a sample of clinical-trial protocols. Finally, the macroeconomic impact, measured in terms of value-added and jobs created by the conducted ISCTs, was calculated using Input–Output analysis by applying an extended Leontief-model.Results:The study demonstrated that €116.22 million spent in ISCTs generated a total value added of €144 million, €74 million direct, in 2018. Each year a medical treatment value of €100 million was financed through 463 ISCTs, with an average value of medical treatment of €37,068 per recruited patient. This represents a significant 0.3% of annual current health-expenditures. In summary, each Euro invested by the pharmaceutical industry in ISCTs generates €1.95 for the Austrian economy. ISCTs also created and secured employment in the extent of 2,021 full-time-equivalents, thus resulting in an employment multiplier of 1.66.Conclusions:In conclusion, conducting clinical-trials by pharmaceutical industry—beside its importance in its own domain—results in tangible benefits and a positive macroeconomic impact that contribute to the sustainability of the Austrian healthcare system by complementing its limited resources. Furthermore, it is a non-negligible factor in locational and industrial policy.

To examine the performance of automated convolutional neuronal network cartilage segmentation in knees with radiographic osteoarthritis (ROA), and its dependence on the OA status of the training set and MRI sequences.

Breath composition is altered in liver diseases. We tested if ion–molecule-reaction mass spectrometry (IMR-MS) combined with a new statistical modality improves the diagnostic accuracy of breath analysis in liver diseases. We analysed 114 molecules in the breath of 126 individuals (healthy controls, and patients with non-alcoholic and alcoholic fatty liver disease and liver cirrhosis) by IMR-MS. Characteristic exhalation patterns were identified for each group. Combining two to seven molecules in the new stacked feature ranking model reached a diagnostic accuracy (area under the curve) for individual liver diseases between 0.88 and 0.97. IMR-MS followed by sophisticated statistical analysis is a promising tool for liver diagnostics by breath analysis.

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases, neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816–induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore, midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally, a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression, or by BH3-mimetics such as obatoclax, may be an attractive therapy concept in SM.

Recent data suggest that the signal transducer and activator of transcription (STAT)5 contributes to differentiation and growth of mast cells. It has also been described that constitutively phosphorylated STAT5 (pSTAT5) plays a pro-oncogenic role in various myeloid neoplasms. We examined the expression of pSTAT5 in neoplastic mast cells in systemic mastocytosis and asked whether the disease-related oncoprotein KIT D816V is involved in STAT5 activation. As assessed by immunohistochemistry using the anti-pSTAT5 antibody AX1, neoplastic mast cells were found to display pSTAT5 in all SM patients examined (n= 40). Expression of pSTAT5 was also demonstrable in the KIT D816V-positive mast cell leukemia cell line HMC-1. Using various staining-protocols, pSTAT5 was found to be located in both the cytoplasmic and nuclear compartment of mast cells. To define the functional role of KIT D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of KIT D816V were used. In these cells, induction of KIT D816V resulted in an increased expression of pSTAT5 without substantial increase in total STAT5. Moreover, the KIT D816V-targeting kinase-inhibitor PKC412 was found to counteract expression of pSTAT5 in HMC-1 cells as well as doxycycline-induced expression of pSTAT5 in Ba/F3 cells. Finally, a dominant negative STAT5-construct was found to inhibit growth of HMC-1 cells. Together, our data show that neoplastic mast cells express cytoplasmic and nuclear pSTAT5, that KIT D816V promotes STAT5-activation, and that STAT5-activation contributes to growth of neoplastic mast cells.

Lung cancer accounts for the most cancer-related deaths. The identification of cancer-associated genes and the related pathways are essential to prevent many types of cancer. In this paper, a more systematic approach is considered. First, we did pathway analysis using Hyper Geometric Distribution (HGD) and significantly overrepresented sets of reactions were identified. Second, feature-selection-based Particle Swarm Optimisation (PSO), Information Gain (IG) and the Biomarker Identifier (BMI) for the identification of different types of lung cancer were used. We also evaluated PSO and developed a new method to determine the BMI thresholds to prioritise genes. We were able to identify sets of key genes that can be found in several pathways. Experimental results show that our method simplifies features effectively and obtains higher classification accuracy than the other methods from the literature.

Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ABL+ human leukemic cells express PI3Kδ and therefore explored its impact on leukemia development. Using PI3Kδ-deficient mice, we define a dual role of PI3Kδ in leukemia. We observed a growth-promoting effect in tumor cells and an essential function in natural killer (NK) cell–mediated tumor surveillance: Abelson-transformed PI3Kδ-deficient cells induced leukemia in RAG2-deficient mice with an increased latency, indicating that PI3Kδ accelerated leukemia progression in vivo. However, the absence of PI3Kδ also affected NK cell–mediated tumor surveillance. PI3Kδ-deficient NK cells failed to lyse a large variety of target cells because of defective degranulation, as also documented by capacitance recordings. Accordingly, transplanted leukemic cells killed PI3Kδ-deficient animals more rapidly. As a net effect, no difference in disease latency in vivo was detected if both leukemic cells and NK cells lack PI3Kδ. Other tumor models confirmed that PI3Kδ-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3Kδ in the NK compartment is as relevant to survival of the mice as the delayed tumor progression. This dual function must be taken into account when using PI3Kδ inhibitors as antileukemic agents in clinical trials.

In this paper, a cellular automaton framework for processing the spatiotemporal spread of infectious diseases is presented. The developed environment simulates and visualizes how infectious diseases might spread, and hence provides a powerful instrument for health care organizations to generate disease prevention and contingency plans. In this study, the outbreak of an avian flu like virus was modeled in the state of Tyrol, and various scenarios such as quarantine, effect of different medications on viral spread and changes of social behavior were simulated.The proposed framework is implemented using the programming language Java. The set up of the simulation environment requires specification of the disease parameters and the geographical information using a population density colored map, enriched with demographic data.The results of the numerical simulations and the analysis of the computed parameters will be used to get a deeper understanding of how the disease spreading mechanisms work, and how to protect the population from contracting the disease. Strategies for optimization of medical treatment and vaccination regimens will also be investigated using our cellular automaton framework.In this study, six different scenarios were simulated. It showed that geographical barriers may help to slow down the spread of an infectious disease, however, when an aggressive and deadly communicable disease spreads, only quarantine and controlled medical treatment are able to stop the outbreak, if at all.

Dasatinib is a multitargeted drug that blocks several tyrosine kinases. Apart from its well-known antileukemic activity, the drug has attracted attention because of potential immunosuppressive and anti-inflammatory effects. We report that dasatinib at 1 μM completely blocks anti-IgE–induced histamine release in blood basophils in healthy donors, and allergen-induced release of histamine in sensitized individuals. In addition, dasatinib inhibited FcϵRI-mediated release of IL-4 and IgE-mediated up-regulation of CD13, CD63, CD164, and CD203c in basophils. The effects of dasatinib were dose-dependent (IC50: 50-500 nM) and specific for FcϵRI activation in that the drug failed to inhibit C5a-induced or Ca-ionophore–induced histamine release. Interestingly, at lower concentrations, dasatinib even promoted FcϵRI-dependent histamine release in basophils in allergic subjects. In consecutive studies, dasatinib was found to interact with and block several FcϵRI downstream targets in basophils, in-cluding Btk. Correspondingly, FcϵRI-mediated histamine secretion in basophils was markedly reduced in Btk knockout mice and in a patient with Btk deficiency. However, the remaining “low-level” mediator secretion in Btk-deficient cells was fully blocked down again by 1 μM dasatinib. Together, these data suggest that dasatinib inhibits FcϵRI-mediated activation of basophils through multiple signaling molecules including Btk. Dasatinib may be an interesting agent for immunologic disorders involving Btk-dependent responses or/and FcϵRI activation of basophils.

The article offers an analysis concerning the importance of tourism and human rights in theological education, with reference from the story of Sodom and Gomorra in the book of Genesis. It contends that the early motivation for tourism was religious pilgrimage but it was accompanied by other motivations including economic interest, military interventions, and the wish to exchange and bring something to other people in the world. A socio-historial exegesis explains that the message of God's justice repeatedly requests people to address the violation of human rights. Moreover, it suggests that theological activities relating to travel must be extended apart from ministry to holidaymakers, prayers for travelers, and to deal with the shadow side of the modern tourism.

In newborn errors of metabolism, biomarkers are urgently needed for disease screening, diagnosis, and monitoring of therapeutic interventions. This article describes a 2-step approach to discovermetabolic markers, which involves (1) the identification ofmarker candidates and (2) the prioritization of thembased on expert knowledge of diseasemetabolism. For step 1, the authors developed a new algorithm, the biomarker identifier (BMI), to identifymarkers fromquantified diseased versus normal tandemmass spectrometry data sets. BMI produces a ranked list ofmarker candidates and discards irrelevant metabolites based on a quality measure, taking into account the discriminatory performance, discriminatory space, and variance ofmetabolites’ concentrations at the state of disease. To determine the ability of identified markers to classify subjects, the authors compared the discriminatory performance of several machine-learning paradigms and described a retrieval technique that searches and classifies abnormal metabolic profiles from a screening database. Seven inborn errors of metabolism— phenylketonuria (PKU), glutaric acidemia type I (GA-I), 3-methylcrotonylglycinemia deficiency (3-MCCD), methylmalonic acidemia (MMA), propionic acidemia (PA), medium-chain acylCoAdehydrogenase deficiency (MCADD), and 3-OH longchain acyl CoA dehydrogenase deficiency (LCHADD)—were investigated. All primarily prioritized marker candidates could be confirmed by literature. Somenovel secondary candidateswere identified (i.e., C16:1 andC4DCfor PKU, C4DCfor GA-I, and C18:1 forMCADD), which require further validation to confirmtheir biochemical role during health and disease.

Cytotoxic hydroxyethyl‐substituted (amine)platinum(II) and ‐(IV) complexes have recently attracted attention because of the ability of their hydroxyethyl groups to coordinate through the oxygen atom to the platinum centre during oxidation with hydrogen peroxide or through intramolecular ligand exchange reactions in dichloroplatinum(II) complexes. The last point in particular is of great interest, because the intramolecular attack of the hydroxy group dramatically influences the mode of action of platinum(II) compounds. On the other hand, there is also the chance to use such reactions specifically for the synthesis of novel anticancer platinum‐based drugs for chemotherapy. We have therefore focused our chemistry program on the synthesis of dichloroplatinum(II) complexes that are in a position to form singly and, especially, doubly ring‐closed alcoholato species and on investigation of their structures by X‐ray crystallography. It was possible to determine the crystal structures of [Pt{(R)‐(−)‐HL}2Cl2], [Pt{(S)‐(+)‐HL}2Cl2], [Pt{(R)‐(−)‐HL}{(S)‐(+)‐HL}Cl2], [Pt{(S)‐(+)‐HL}{(S)‐(+)‐L}Cl], [Pt{(R)‐(−)‐L}2] and [Pt{(S)‐(+)‐L}2] (HL = 2‐aminobutanol‐κN, L = 2‐aminobutanolato‐κ2N,O). The results obtained may represent the first step towards novel prodrugs for platinum‐based antitumour chemotherapy. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

The present study has focused on a general reaction procedure for the derivatization of hydroxyethyl‐substituted tetrachloro(ethane‐1,2‐diamine)platinum(IV) compounds at peripheral hydroxyl groups using acyl chlorides. A new class of platinum(IV) complexes could be synthesized which now opens the possibility for the first time to couple the cytotoxic platinum(IV) moiety to carrier molecules like proteins and antibodies in a very selective way. Moreover, it is now possible to synthesize platinum(IV) complexes with ligands in the equatorial plane which are not very stable in the presence of oxidizing agents.

The bis(ethanolamine)platinum complexes (SP‐4‐2)‐dichlorobis(2‐hydroxyethylamine)platinum(II) (1) and (OC‐6‐22)‐tetrachlororbis(2‐hydroxyethylamine)platinum(IV) (2) have been synthesized and their chemistry in aqueous solution has been investigated due to the fact that 1forms very stable monoadducts with 5′‐GMP. In water 1and 2are converted into (SP‐4‐3)‐chloro(2‐ethanolatoamine‐κ2N,O)(2‐hydroxyethylamine)platinum(II) (3) and (OC‐6‐31)‐trichloro(2‐ethanolatoamine‐κ2N,O)(2‐hydroxyethylamine)platinum(IV) (4) with a chelating ethanolatoamine ligand by a intramolecular ligand exchange reaction, which was confirmed by 1H and 2D 15N,1H‐HMQC NMR experiments and the crystal structure determination of 4.

Hematopoietic stem cells (HSCs) sustain hematopoiesis throughout life. HSCs exit dormancy to restore hemostasis in response to stressful events, such as acute blood loss, and must return to a quiescent state to prevent their exhaustion and resulting bone marrow failure. HSC activation is driven in part through the phosphatidylinositol 3-kinase (PI3K)/AKT/mTORC1 signaling pathway, but less is known about the cell-intrinsic pathways that control HSC dormancy. Here, we delineate an ERK-dependent, rate-limiting feedback mechanism that controls HSC fitness and their re-entry into quiescence. We show that the MEK/ERK and PI3K pathways are synchronously activated in HSCs during emergency hematopoiesis and that feedback phosphorylation of MEK1 by activated ERK counterbalances AKT/mTORC1 activation. Genetic or chemical ablation of this feedback loop tilts the balance between HSC dormancy and activation, increasing differentiated cell output and accelerating HSC exhaustion. These results suggest that MEK inhibitors developed for cancer therapy may find additional utility in controlling HSC activation.

Myelodysplastic syndromes (MDS) are a group of clonal myeloid neoplasms characterized by ineffective erythropoiesis, peripheral cytopenia(s), and an increased risk to transform to secondary acute myeloid leukemia (AML). The prognosis in MDS is variable and depends on the variant of disease, other disease-related features, and patient-related parameters. In the present study, the influence of comorbidity on survival and AML evolution was analyzed retrospectively in 582 patients (270 females and 312 males, f/m ratio: 1:1.2) with de novo MDS (observation period: 1985–2007). The median age was 71 years (range 18–96 years). Of the 582 patients, 275 died so far. The median survival (OS) of all patients was 3.12 years, and the median event-free survival (EFS) was 2.3 years. The median AML-free survival (AFS) was not reached. All in all, 127 patients (22%) developed secondary AML after a median time of 9.7 months (range 0.3–116.6 months). Two different scoring systems for comorbidity, the hematopoietic stem cell transplantation comorbidity index (HCT-CI) and the Charlson comorbidity index (CCI) were applied. As assessed by log rank test, the overall survival (OS) was found to differ among patients in the three different HCT-CI risk groups (p<0.05) and among patients in the four different CCI risk groups. By univariate analysis, the HCT-CI was found to be of prognostic value for OS and EFS in patients meeting WHO- or FAB criteria (p<0.05). The CCI was also found to be of prognostic value for OS in patients diagnosed according to either WHO or FAB criteria (p<0.05). With regard to EFS, the CCI was a prognostically significant variable only for patients meeting WHO criteria (p<0.05), but not in patients diagnosed according to FAB criteria (p>0.05). Calculating AML-free survival (AFS), neither the CCI nor the HCT-CI were of prognostic significance (p>0.05). To evaluate whether comorbity is an independent prognostic parameter in patients with MDS, multivariate analyses were performed. These analyses included the HCT-CT or the CCI together with IPSS, LDH, and the patients’ age. In these analyses, chronic comorbid conditions were found to be independent prognostic risk factors concerning OS and EFS, but not concerning AFS. Specifically, the HCT-CI was an independent prognostic parameter regarding OS (p<0.05) and EFS (p<0.05) for patients diagnosed according to WHO- or FAB-criteria. In contrast, the CCI was of prognostic significance regarding OS for patients meeting WHO- or FAB-criteria, whereas the CCI was not found to be an independent prognostic factor regarding EFS (p>0.05). Regardless of the score applied (HCT-CI or CCI), the highest predictive value of comorbidity was observed in IPSS low risk patients (p<0.05) concerning OS. Of the other variables included in our multivariate analysis, the IPSS was an independent prognostic parameter for OS, EFS, and AFS. Interestingly, age was an independent prognostic variable for OS and EFS, but not concerning AFS, similar to the impact of comorbidity, whereas LDH was an independent predictive factor concerning EFS and AFS. Together, our data show that comorbidity is an independent risk factor for survival in patients with MDS. Therefore, comorbidity should be considered as an important co-variable in the risk assessment in MDS and in the overall treatment plan in these patients.

Chronic myeloid leukemia (CML) is a stem cell disease characterized by the BCR/ABL oncoprotein. The ABL kinase inhibitor imatinib is effective in most patients and considered standard first line therapy. However, not all patients show a long-lasting response. Treatment failure is usually associated with the occurrence of imatinib-resistant mutants of BCR/ABL. For these patients, novel multi-kinase inhibitors such as dasatinib represent alternative treatment options. Still, however, not all patients respond to these drugs, especially when leukemic cells bear the BCR/ABL mutant T315I that confers resistance against most kinase-blockers. Bosutinib is a novel multi-kinase inhibitor that has been described to act growth-inhibitory in ABL-transformed leukemias. In the current study, we examined the effects of bosutinib alone and in combination with dasatinib on growth and survival of CML cells. Bosutinib was found to inhibit 3H-thymidine uptake and thus proliferation in imatinib-sensitive and imatinib-resistant K562 cells in a dose-dependent manner, with identical IC50 values (10–100 nM). Moreover, bosutinib was found to inhibit the growth of primary CML cells and Ba/F3 cells bearing various imatinibresistant mutants of BCR/ABL, except the T315I mutant (IC50>1 μM). The growth-inhibitory effects of bosutinib were found to be associated with signs of apoptosis. Dasatinib showed similar effects on CML cells, and again did not block the growth of subclones bearing BCR/ABL T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth inhibition in primary CML cells exhibiting BCR/ABL T315I at pharmacologic concentrations (0.01–1 μM). Clear synergistic effects were also observed in imatinib-sensitive and imatinib-resistant K562 cells as well as in Ba/F3 cells bearing BCR/ABL T315I. In parallel, we performed multiplexed kinase assays as well as chemical proteomics analysis and mass spectrometry using K562 cells and primary CML cells and coupleable dasatinib and bosutinib analogues. In these experiments, dasatinib and bosutinib were found to express an overlapping, but non-identical profile of target kinases. As expected, both drugs were found to bind to wt ABL, SRC kinases, and TEC-family kinases including BTK. Specific targets preferentially bound and inhibited by bosutinib were STE20s, the FES/FER family, CAMKIIG, PYK2 and TBK1. We were also able to confirm that the dasatinib-targets KIT and PDGFRA are not recognized by bosutinib. Interestingly, whereas wt ABL (IC50<0.5 nM) and most of the ABL mutants tested (H396P, M351T, Q252H, and Y253F) were all completely inhibited by both drugs at 1 μM in the kinase assay, the ABL T315I mutant was inhibited by bosutinib (IC50=26 nM) almost 70 times more potently than by dasatinib. Together, these data show that bosutinib and dasatinib synergize with each other in producing antileukemic effects on CML cells including BCR/ABL T315I+ subclones. These synergistic effects may be explained by differential target kinase profiles and by the fact that bosutinib retains some activity against the BCR/ABL T315I mutant kinase.

The malignant clone in acute myeloid leukemia (AML) is organized hierarchically with more mature cells programmed to undergo natural apoptosis after a variable number of cell divisions and immature primitive cells that have self-renewal and NOD/SCID mouse-repopulating capacity. In most types of AML, leukemic stem cells supposedly reside within the CD34+/CD38− fraction of the leukemic clone. Because of their long term leukemia-repopulating capacity, AML stem cells are a logic target of therapy. However, so far, little is known about the regulation of growth and survival of these cells. We examined the expression of cytokine receptors (SCFR/KIT, IL-3Rα, GM-CSFRα, IL-3/GM-CSFRβ, G-CSFR, M-CSFR, TGFβR/endoglin, EPOR, TPOR/MPL, FLT3, VEGFR/KDR) and of other molecular targets and markers (CD33, CD44, CD133) on CD34+/CD38− cells in patients with AML (n=30), and determined responses to cytokine-ligands, conventional antileukemic drugs (ARA-C, fludarabine) or targeted drugs (Gemtuzumab/Ozogamicin, GO = Mylotarg®) in these cells. Apoptosis in AML stem cells was analyzed by combined staining for surface markers and AnnexinV. In a group of patients, CD34+/CD38- cells were purified to homogeneity by cell sorting and examined for 3H-thymidine uptake. Unexpectedly, AML stem cells were found to display a highly variable pattern of cytokine receptors and cell surface targets. In fact, only the IL-3R and CD44 were expressed consistently on all AML stem cells in all donors tested. In most patients, at least a subset of AML stem cells also co-expressed the SCFR/KIT, G-CSFR, TGFβR, FLT3, CD33, and CD133. By contrast, AML stem cells in most donors were found to lack substantial amounts of the GM-CSFR, M-CSFR, EPOR, TPOR, and VEGFR/KDR. When cultured in RPMI-1640 medium plus 10% FCS, about 5–15% of the CD34+/CD38- cells and about 10–40% of the more mature CD34+ AML cells were found to undergo spontaneous apoptosis within 48 hours. Spontaneous apoptosis was prevented by exposure to SCF, IL-3, or G-CSF, but not by exposure to EPO. ARA-C (0.5–5 μg/ml), fludarabine (0.1–5 μg/ml), and GO/Mylotarg® (1 μg/ml) were found to promote apoptosis in CD34+/CD38− cells and in more mature AML cells in all donors tested. The effects of ARA-C and fludarabine on AML cells were found to be dose-dependent. Moreover, we were able to show that ARA-C (0.5 μg/ml) and fludarabine (0.1 μg/ml) cooperate with each other in producing apoptosis in AML (stem) cells. 3H-thymidine uptake experiments performed on purified CD34+/CD38− AML stem cells confirmed the growth-inhibitory effects of these drugs. In particular, ARA-C and fludarabine were found to inhibit the cytokine-induced 3H-thymidine uptake in sorted AML stem cells in all donors examined. In summary, our data show that multi-color flow cytometry and combined staining for surface markers and AnnexinV is a powerful approach to determine apoptosis-preventing effects of cytokines and apoptosis-inducing effects of anti-leukemic drugs in immature CD34+/CD38− AML progenitor cells. Using this assay, it should be possible to identify combinations of targeted and/or conventional drugs eliminating maximal numbers of leukemic stem cells in AML.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease in which BCR/ABL enhances growth and survival of leukemic cells. In most patients, the disease can be kept under control by the BCR/ABL tyrosine kinase inhibitor imatinib (STI571; Novartis Basel, Switzerland). However, resistance or intolerance against imatinib may occur during therapy. Therefore, current research is focusing on novel targets and targeted drugs in CML. Polo-like kinase 1 (Plk-1) is a serine/threonine kinase that plays an essential role in mitosis and is expressed in activated/phosphorylated form in various malignancies including acute myeloid leukemia (AML). BI 2536 (Boehringer Ingelheim GmbH, Germany) is a novel selective inhibitor of Plk-1, that is currently tested in AML-trials. In this study, we have evaluated expression and the potential role of Plk-1 as a novel target in CML cells. As assessed by PCR, Plk-1 mRNA was found to be expressed abundantly in primary CML cells and in the CML cell line K562, whereas normal peripheral blood cells did not express detectable levels of Plk-1 mRNA. The Plk-1 protein was detected in primary CML cells and K562 cells by immunocytochemistry. In consecutive experiments, we were able to show that CML cells display phosphorylated Plk-1. As assessed by 3H-thymidine-uptake experiments, BI 2536 was found to inhibit the proliferation of K562 cells in a dose-dependent manner (IC50 5–15 nM). Moreover, BI 2536 was found to inhibit the proliferation of both imatinib-naive (n=6) and imatinib-resistant (n=3) primary CML cells (IC50: 1–15 nM). The growth-inhibitory effect of BI 2536 on CML cells was found to be associated with mitotic arrest, a G2-M cell cycle arrest, and consecutive apoptosis. In normal bone marrow or peripheral blood mononuclear cells, neither mitotic cell arrest nor apoptosis were observed after exposure to BI 2536. In further experiments, primary CML cells were coincubated with BI 2536 plus imatinib or with BI 2536 plus nilotinib (AMN107; Novartis) at fixed ratio of drug concentrations. In these experiments, BI 2536 was found to synergize with both tyrosine kinase inhibitors in counteracting the proliferation of CML cells. In conclusion, our data show that Plk-1 is expressed in activated form in CML cells and plays a role in cell cycle progression and cell viability. Targeting Plk-1 with BI 2536 leads to mitotic arrest, growth inhibition, and apoptosis in imatinib-naive and imatinib-resistant leukemic cells. Moreover, BI 2536 synergizes with imatinib and nilotinib in counteracting the growth of neoplastic cells in CML. Targeting of Plk-1 may be a novel interesting pharmacologic approach to counteract growth of CML cells.

Chronic eosinophilic leukemia (CEL) is a myeloproliferative disorder characterized by molecular and/or cytogenetic evidence of monoclonality of eosinophils, sustained marked eosinophilia, and consecutive organ damage. In a majority of patients with CEL with or without associated mastocytosis, the transforming mutation FIP1L1-PDGFRα and the related CHIC2 deletion is found. The respective oncoprotein, FIP1L1-PDGFRα, is considered to play a major role in malignant cell growth in CEL. The tyrosine kinase (TK) inhibitor imatinib (STI571) has been described to counteract the TK activity of FIP1L1-PDGFRα in most patients, and has been introduced as a novel effective therapy in CEL. However, not all patients with CEL show a response to imatinib. Therefore, several attempts have been made to identify other TK inhibitors that counteract growth of neoplastic eosinophils in CEL. We provide evidence that dasatinib, a multi-targeted kinase inhibitor, blocks the growth and survival of EOL-1, an eosinophil leukemia cell line carrying FIP1L1-PDGFRα. The effects of dasatinib on proliferation of EOL-1 cells were dose-dependent, with an IC50 of 0.5–1 nM, that was found to be in the same range compared to IC50 values produced by imatinib. Dasatinib was also found to induce apoptosis in EOL-1 cells in a dose-dependent manner (IC-50: 1–10 nM). The apoptosis-inducing effects of dasatinib on EOL-1 cells were demonstrable by light microscopy, flow cytometry, and by a Tunel assay. To further examine the mechanism of growth inhibition induced by dasatinib in neoplastic eosinophils, Western blot experiments were performed using antibodies directed against phosphorylated or total PDGFRα. In these experiments, we were able to show that dasatinib at 1 μM completely blocks the phosphorylation of FIP1L1-PDGFRα in EOL-1 cells. In summary, our data show that dasatinib inhibits the growth of leukemic eosinophils through targeting of the TK activity of the disease-related oncoprotein FIP1L1-PDGFRα. Based on this observation, dasatinib may be considered as a new interesting treatment option for patients with CEL. As dasatinib is also known to block various KIT mutants as well as wild type KIT, such therapy may also be of interest for patients who have systemic mastocytosis (SM) with an associated CEL (SM-CEL).

Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC) in various internal organs. In most patients, the D816V-mutated variant of c-KIT, which mediates resistance against several tyrosine kinase (TK) inhibitors like imatinib, is found. In advanced SM, the response of neoplastic MC to conventional drugs is poor and the prognosis is grave. Therefore current research is attempting to identify novel targets in neoplastic MC. Polo-like kinase 1 (Plk-1) is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias. In the present study, we analyzed expression and function of Plk-1 in neoplastic human MC, and asked whether Plk-1 can serve as a target of therapy in SM. As determined by immunohistochemistry, primary neoplastic MC were found to display activated/phosphorylated Plk-1 in all patients examined (n=5). The human MC leukemia cell line HMC-1 was also found to exhibit activated Plk-1. In addition, we found that primary neoplastic MC as well as HMC-1 cells express Plk-1 mRNA in RT-PCR experiments. As assessed by 3H-thymidine-uptake experiments, the Plk-1-targeting drug BI 2536 (Boehringer Ingelheim GmbH, Germany) was found to inhibit the proliferation of HMC-1 cells in a dose-dependent manner (IC50 5–15 nM). The effect of BI 2536 was seen in both subclones of HMC-1, i.e. in HMC-1.1 cells displaying KIT G560V (but not KIT D816V), and HMC-1.2 cells exhibiting both KIT G560V and KIT D816V, with comparable IC50 values. Moreover, BI 2536 was found to inhibit the proliferation of primary neoplastic cells, with IC50 values ranging between 5 and 50 nM. The growth-inhibitory effects of BI 2536 on HMC-1 cells were found to be associated with mitotic arrest and G2-M cell cycle arrest as well as consecutive apoptosis. In normal bone marrow or peripheral blood mononuclear cells, neither mitotic cell arrest nor apoptosis were observed after treatment with BI 2536. In a consecutive phase of the study, we asked whether combined targeting of KIT D816V and Plk-1 would lead to synergistic drug-interactions. For this purpose, HMC-1 cells and primary neoplastic MC were coincubated with BI 2536 and midostaurin (PKC412), a multitargeted kinase inhibitor that blocks KIT D816V TK activity. In these experiments, BI 2536 was found to synergize with midostaurin in counteracting the proliferation of HMC-1 cells and primary neoplastic MC. In conclusion, our data show that activated Plk-1 is detectable in MC neoplasms and plays a role in cell cycle progression and viability of neoplastic MC. Targeting of Plk-1 with BI 2536 leads to growth inhibition and apoptosis in neoplastic MC. Furthermore, BI 2536 synergizes with midostaurin in counteracting growth of neoplastic MC. Targeting of Plk-1 may be an attractive new pharmacologic concept in advanced SM.

Motivation: Classification is an important data mining task in biomedicine. In particular, classification on biomedical data often claims the separation of pathological and healthy samples with highest discriminatory performance for diagnostic issues. Even more important than the overall accuracy is the balance of a classifier, particularly if datasets of unbalanced class size are examined. Results: We present a novel instance-based classification technique which takes both information of different local density of data objects and local cluster structures into account. Our method, which adopts the basic ideas of density-based outlier detection, determines the local point density in the neighborhood of an object to be classified and of all clusters in the corresponding region. A data object is assigned to that class where it fits best into the local cluster structure. The experimental evaluation on biomedical data demonstrates that our approach outperforms most popular classification methods. Availability: The algorithm LCF is available for testing under http://biomed.umit.at/upload/lcfx.zip Contact: christian.baumgartner@umit.at