The flavoprotein FOXRED2 reductively activates nitro-chloromethylbenzindolines and other hypoxia-targeting prodrugs

The nitro-chloromethylbenzindoline prodrug SN29428 has been rationally designed to target tumour hypoxia. SN29428 is metabolised to a DNA minor groove alkylator via oxygen-sensitive reductive activation initiated by unknown one-electron reductases. The present study sought to identify reductases capable of activating SN29428 in tumours. Expression of candidate reductases in cell lines was modulated using forced expression and, for P450 (cytochrome) oxidoreductase (POR), by zinc finger nuclease-mediated gene knockout. Affymetrix microarray mRNA expression of flavoreductases was correlated with SN29428 activation in a panel of 23 cancer cell lines. Reductive activation and cytotoxicity of prodrugs were measured using mass spectrometry and antiproliferative assays, respectively. SN29428 activation under hypoxia was strongly attenuated by the pan-flavoprotein inhibitor diphenyliodonium, but less so by knockout of POR suggesting other flavoreductases contribute. Forced expression of 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), as well as POR, increased activation of SN29428 in hypoxic HCT 116 cells. SN29428 activation strongly correlated with expression of POR and also FAD-dependent oxidoreductase domain containing 2 (FOXRED2), in cancer cell lines. This association persisted after removing the effect of POR enzyme activity using first-order partial correlation. Forced expression of FOXRED2 increased SN29428 activation and cytotoxicity in hypoxic HEK293 cells and also increased activation of hypoxia-targeted prodrugs PR-104A, tirapazamine and SN30000, and increased cytotoxicity of the clinical-stage prodrug TH-302. Thus this study has identified three flavoreductases capable of enzymatically activating SN29428, one of which (FOXRED2) has not previously been implicated in xenobiotic metabolism. These results will inform future development of biomarkers predictive of SN29428 sensitivity.     

Iron Deficiency Is Associated With Impaired Biventricular Reserve and Reduced Exercise Capacity in Patients With Unexplained Dyspnea

BackgroundIron deficiency (ID) is frequent and associated with diminished exercise capacity in heart failure (HF), but its contribution to unexplained dyspnea without a HF diagnosis at rest remains unclear.Methods and ResultsConsecutive patients with unexplained dyspnea and normal echocardiography and pulmonary function tests at rest underwent prospective standardized cardiopulmonary exercise testing with echocardiography in a tertiary care dyspnea clinic. ID was defined as ferritin of <300 µg/L and a transferrin saturation of <20% and its impact on peak oxygen uptake (peakVO₂), biventricular response to exercise, and peripheral oxygen extraction was assessed. Of 272 patients who underwent cardiopulmonary exercise testing with echocardiography, 63 (23%) had ID. For a similar respiratory exchange ratio, patients with ID had lower peakVO₂ (14.6 ± 7.6 mL/kg/minvs 17.8 ± 8.8 mL/kg/min; P = .009) and maximal workload (89 ± 50 watt vs 108 ± 56 watt P = .047), even after adjustment for the presence of anemia. At rest, patients with ID had a similar left ventricular and right ventricular (RV) contractile function. During exercise, patients with ID had lower cardiac output reserve (P < .05) and depressed RV function by tricuspid s' (P = .004), tricuspid annular plane systolic excursion (P = .034), and RV end-systolic pressure-area ratio (P = .038), with more RV–pulmonary artery uncoupling measured by tricuspid annular plane systolic excursion/systolic pulmonary arterial pressure ratio (P = .023). RV end-systolic pressure-area ratio change from rest to peak exercise, as a load-insensitive metric of RV contractility, was lower in patients with ID (2.09 ± 0.72 mm Hg/cm² vs 2.58 ± 1.14 mm Hg/cm²; P < .001). ID was associated with impaired peripheral oxygen extraction (peakVO₂/peak cardiac output; P = .036). Cardiopulmonary exercise testing with echocardiography resulted in a diagnosis of HF with preserved ejection fraction in 71 patients (26%) based on an exercise E/e' ratio of >14, with equal distribution in patients with (28.6%) or without ID (25.4%, P = .611). None of these findings were influenced in a sensitivity analysis adjusted for a final diagnosis of HFpEF as etiology for the unexplained dyspnea.ConclusionsIn patients with unexplained dyspnea without clear HF at rest, ID is common and associated with decreased exercise capacity, diminished biventricular contractile reserve, and decreased peripheral oxygen extraction.     

Pyrido[4,3-e][1,2,4]triazolo[4,3-a]pyrazines as Selective,Brain Penetrant Phosphodiesterase 2 (PDE2) Inhibitors

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Randomized comparison of low-dose versus high-dose interferon-alfa in chronic myeloid leukemia: prospective collaboration of 3 joint trials by the MRC and HOVON groups

The optimal dose of interferon-alfa (IFN) for chronic myeloid leukemia (CML) is unknown. Retrospective analyses suggest that low doses are as effective as high doses, with less toxicity and fewer patients abandoning the drug. The Dutch Hemato-Oncology Association (HOVON) and British Medical Research Council (MRC) cooperative groups jointly performed randomized trials in newly diagnosed CML patients, comparing high-dose IFN (5 MIU/m(2) daily) with low-dose (3 MIU, 5 times a week). Both arms allowed additional hydroxyurea to keep the white blood cell count lower than 5 x 10(9)/L. Quality of life data were collected in a subset of patients. Between 1993 and 2001, 407 patients were randomized. At a median follow-up of 53 months, there were no significant differences in overall survival (odds ratio = 1.09, 95% confidence interval, 0.81-1.46), progression-free survival, and complete hematologic or major cytogenetic responses. Fewer patients in the low-dose group abandoned IFN for reasons other than transplant or progressive disease (P =.002, 58% vs 72% at 5 years). Quality of life data showed comparable results in both arms for most factors. There is no evidence of benefit for high-dose IFN compared with low-dose for the treatment of CML. Therefore, when IFN is combined with other drugs, low-dose IFN is advised, to minimize toxicity and costs.     

Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen-specific cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs) specific for hematopoietic-restricted minor histocompatibility antigens (mHags) are important reagents for adoptive immunotherapy of relapsed leukemia after allogeneic stem cell transplantation. However, expansion of these CTLs to therapeutic numbers is often hampered by the limited supply of antigen-presenting cells (APCs). Therefore, we evaluated whether cell-sized latex beads coated with HLA/mHag complexes HLA-A2/HA-1 or HLA-A2/HA-2 and recombinant CD80 and CD54 molecules can replace professional APCs. The artificial antigen-presenting constructs (aAPCs) effectively stimulated HA-1– and HA-2–specific CTL clones as shown by ligand-specific expansion, cytokine production, and maintenance of cytotoxic activity, without alteration of CTL phenotype. Furthermore, HA-1–specific polyclonal CTL lines were enriched as efficiently by aAPCs as by autologous HA-1 peptide-pulsed dendritic cells. Thus, aAPCs coated with HLA/mHag complexes, CD80, and CD54 may serve as tools for in vitro enrichment of immunotherapeutic mHag-specific CTL lines.      

The origin of secondary microseism Love waves

The interaction of ocean surface waves produces pressure fluctuations at the seafloor capable of generating seismic waves in the solid Earth. The accepted mechanism satisfactorily explains secondary microseisms of the Rayleigh type, but it does not justify the presence of transversely polarized Love waves, nevertheless widely observed. An explanation for two-thirds of the worldwide ambient wave field has been wanting for over a century. Using numerical simulations of global-scale seismic wave propagation at unprecedented high frequency, here we explain the origin of secondary microseism Love waves. A small fraction of those is generated by boundary force-splitting at bathymetric inclines, but the majority is generated by the interaction of the seismic wave field with three-dimensional heterogeneity within the Earth. We present evidence for an ergodic model that explains observed seismic wave partitioning, a requirement for full-wave field ambient-noise tomography to account for realistic source distributions.

The surface of the Earth is continuously subjected to perturbing forces that generate seismic waves. Given that 70% of the surface of our planet is covered by oceans, seismic signals due to ocean storms represent the vast majority of seismic data recorded by seismometers on Earth (1). Such data carry information about the energy exchange between different Earth systems, allowing for probing our changing climate (2–4) as well as imaging the internal structure of the Earth (5). The strongest vibrations are called secondary microseisms, excited in the 0.1 to 0.3 Hz frequency range by nonlinear ocean wave–wave interaction (6, 7). They are predominantly composed of seismic surface waves, and Rayleigh waves dominate the vertical component of microseism records (8).The generation mechanism currently accepted for secondary microseisms explains the Rayleigh wave content of vertical-component noise records (9). Secondary microseisms are produced by pressure-like sources at the surface of the ocean. Rayleigh waves are excited below the seafloor due to constructive interference of P and SV body waves. At the ocean–crust interface, they are called Scholte waves when their phase velocity becomes smaller than the minimum phase velocity of the system (10). While at longer periods, ocean waves can directly couple with the seafloor and generate Love waves (11, 12), the generation mechanism of secondary microseisms cannot explain the presence of Love waves on the horizontal components of microseismic records. Observations of secondary microseism Love waves date back to the early (13) and middle (14) 20th century. A few recent studies based on high-quality digital data focused on quantifying the Love-to-Rayleigh ratio in the secondary microseism frequency range (SI Appendix, Table S1). They found that Love-to-Rayleigh ratios are frequency dependent (15) and show a predominance of Rayleigh waves (16, 17), with few exceptions (18).Hypotheses for the generation of secondary microseism Love waves envisage that they can be generated either in the region where the pressure power spectral density (PSD) is strong or along distinct propagation paths within the Earth. The first hypothesis is supported by the presence of bathymetric inclines in the source regions. Such bathymetry may lead to splitting of the vertical second-order pressure force in a component perpendicular to inclines—responsible for Rayleigh waves—and a component tangent to inclines—responsible for Love waves. The second hypothesis is supported by the presence of lateral heterogeneities within the Earth, which can lead to the generation of Love waves due to scattering and focusing/defocusing effects. Ref. 8 observed Love and Rayleigh waves coming from the same direction, concluding that Love waves do originate in the source region. On the other hand, ref. 19 noted that the greater the distance of propagation of Rayleigh waves, the larger the Love wave energy. In addition to these hypotheses, Love waves may originate from Rayleigh-to-Love wave conversion at the ocean–continent boundary, although early numerical simulations suggest that only a few percent of incident Rayleigh wave energy can be converted to Love wave energy (20). To date, no comprehensive theoretical investigations as to which mechanisms can lead to the observed secondary microseism Love waves have been conducted.     

Minor histocompatibility antigens in human stem cell transplantation

Minor histocompatibility antigens (mHags) play a major role in graft rejection, the induction of detrimental graft-vs-host disease (GVHD), and the development of the beneficial graft-vs-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT). mHags can be defined as amino acid polymorphisms in cellular proteins that can lead to differential presentation of antigenic peptides in HLA molecules and therefore to differential recognition by T cells. The tissue distribution of the mHags and the HLA molecules by which they can be presented play a significant role in the clinical outcome of T-cell responses against these antigens. In part, differential recognition by T cells of mHags specifically expressed in hematopoietic cells, including the malignant cells from the recipient may result in GVL reactivity without concurrent GVHD. Furthermore, T-cell responses against proteins solely expressed in hematopoietic cell lineages from which the malignancy is derived may be appropriate mediators of GVL reactivity without GVHD induction. Characterization of clinical immune responses in patients treated for relapsed leukemia after allogeneic SCT with donor lymphocyte infusion in the absence of GVHD may lead to the characterization of new mHags that can be exploited to generate tumor-specific immune responses. By in vitro generation of T-cell responses against defined mHags, the efficacy and specificity of cellular immunotherapy against hematologic malignancies in the context of allogeneic transplantation may be improved.