Decitabine enhances anti-CD33 monoclonal antibody BI 836858-mediated natural killer ADCC against AML blasts.

Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

The interleukin-3 receptor CD123 targeted SL-401 mediates potent cytotoxic activity against CD34+CD123+ cells from acute myeloid leukemia/myelodysplastic syndrome patients and healthy donors.

Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123- lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a "bridge-to-transplant" before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.

CD36-dependent 7-ketocholesterol accumulation in macrophages mediates progression of atherosclerosis in response to chronic air pollution exposure.

RATIONALE: Air pollution exposure has been shown to potentiate plaque progression in humans and animals. Our previous studies have suggested a role for oxidized lipids in mediating adverse vascular effect of air pollution. However, the types of oxidized lipids formed in response to air pollutants and how this occurs and their relevance to atherosclerosis are not fully understood. OBJECTIVE: To investigate the mechanisms by which particulate matter <2.5 µm (PM2.5) induces progression of atherosclerosis. METHODS AND RESULTS: Atherosclerosis-prone ApoE(-/-) or LDLR(-/-) mice were exposed to filtered air or concentrated ambient PM2.5 using a versatile aerosol concentrator enrichment system for 6 months. PM2.5 increased 7-ketocholesterol (7-KCh), an oxidatively modified form of cholesterol, in plasma intermediate density lipoprotein/low-density lipoprotein fraction and in aortic plaque concomitant with progression of atherosclerosis and increased CD36 expression in plaque macrophages from PM2.5-exposed mice. Macrophages isolated from PM2.5-exposed mice displayed increased uptake of oxidized lipids without alterations in their efflux capacity. Consistent with these finding, CD36-positive macrophages displayed a heightened capacity for oxidized lipid uptake. Deficiency of CD36 on hematopoietic cells diminished the effect of air pollution on 7-KCh accumulation, foam cell formation, and atherosclerosis. CONCLUSIONS: Our results suggest a potential role for CD36-mediated abnormal accumulations of oxidized lipids, such as 7-KCh, in air pollution-induced atherosclerosis progression.

α-Gal Nanoparticles in CNS Trauma: I. In Vitro Activation of Microglia Towards a Pro-Healing State.

BACKGROUND: Macrophages and microglia play critical roles after spinal cord injury (SCI), with the pro-healing, anti-inflammatory (M2) subtype being implicated in tissue repair. We hypothesize that promoting this phenotype within the post-injured cord microenvironment may provide beneficial effects for mitigating tissue damage. As a proof of concept, we propose the use of nanoparticles incorporating the carbohydrate antigen, galactose-α-1,3-galactose (α-gal epitope) as an immunomodulator to transition human microglia (HMC3) cells toward a pro-healing state. METHODS: Quiescent HMC3 cells were acutely exposed to α-gal nanoparticles in the presence of human serum and subsequently characterized for changes in cell shape, expression of anti or pro-inflammatory markers, and secretion of phenotype-specific cytokines. RESULTS: HMC3 cells treated with serum activated α-gal nanoparticles exhibited rapid enlargement and shape change in addition to expressing CD68. Moreover, these activated cells showed increased expression of anti-inflammatory markers like Arginase-1 and CD206 without increasing production of pro-inflammatory cytokines TNF-α or IL-6. CONCLUSION: This study is the first to show that resting human microglia exposed to a complex of α-gal nanoparticles and anti-Gal (from human serum) can be activated and polarized toward a putative M2 state. The data suggests that α-gal nanoparticles may have therapeutic relevance to the CNS microenvironment, in both recruiting and polarizing macrophages/microglia at the application site. The immunomodulatory activity of these α-gal nanoparticles post-SCI is further described in the companion work (Part II).

α-Gal Nanoparticles in CNS Trauma: II. Immunomodulation Following Spinal Cord Injury (SCI) Improves Functional Outcomes.

BACKGROUND: Previous investigations have shown that local application of nanoparticles presenting the carbohydrate moiety galactose-α-1,3-galactose (α-gal epitopes) enhance wound healing by activating the complement system and recruiting pro-healing macrophages to the injury site. Our companion in vitro paper suggest α-gal epitopes can similarly recruit and polarize human microglia toward a pro-healing phenotype. In this continuation study, we investigate the in vivo implications of α-gal nanoparticle administration directly to the injured spinal cord. METHODS: α-Gal knock-out (KO) mice subjected to spinal cord crush were injected either with saline (control) or with α-gal nanoparticles immediately following injury. Animals were assessed longitudinally with neurobehavioral and histological endpoints. RESULTS: Mice injected with α-gal nanoparticles showed increased recruitment of anti-inflammatory macrophages to the injection site in conjunction with increased production of anti-inflammatory markers and a reduction in apoptosis. Further, the treated group showed increased axonal infiltration into the lesion, a reduction in reactive astrocyte populations and increased angiogenesis. These results translated into improved sensorimotor metrics versus the control group. CONCLUSIONS: Application of α-gal nanoparticles after spinal cord injury (SCI) induces a pro-healing inflammatory response resulting in neuroprotection, improved axonal ingrowth into the lesion and enhanced sensorimotor recovery. The data shows α-gal nanoparticles may be a promising avenue for further study in CNS trauma.

Use of weak DC electric fields to rapidly align mammalian cells.

Objective.The ability to modulate cell morphology has clinical relevance in regenerative biology. For example, cells of the skeletal muscle, peripheral nerve and vasculature have specific oriented architectures that emerge from unique structure-function relationships. Methods that can induce similar cell morphologiesin vitrocan be of use in the development of biomimetic constructs for the repair or replacement of damaged tissues. In this work, we demonstrate that direct current (DC) electric fields (EFs) can be used as a tool to globally align cell populationsin vitro. Approach.Using a 2D culture chamber system, we were able to quickly (within hours) align Schwann cells at different culture densities with an application of steady EFs at 200-500 mV mm-1.Main results.Cellular alignment was perpendicular to the field vector and varied proportionately as a function of field magnitude. In addition, the degree of cellular alignment was also dependent on cellular density. Even well-established Schwann cell monolayers were responsive to the applied DC fields with cells retracting parallel oriented processes (with respect to the imposed field) and re-extending them along the perpendicular axis. When the DC field was removed, monolayers retained the aligned morphology for many days afterwards, likely due to contact inhibition. We further show the method is applicable to other field-responsive cells, such as 3T3 fibroblasts.Significance.The patterned cells provided nanoscale haptotactic cues and can be subsequently used as a basal layer for co-culturing or manipulated for other applications. DC fields represent a rapid, simple, and efficient technique compared to other cell patterning methods such as substrate manipulation.

Polo-like kinase inhibitor volasertib marginally enhances the efficacy of the novel Fc-engineered anti-CD33 antibody BI 836858 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the second most common type of leukemia in adults. Incidence of AML increases with age with a peak incidence at 67 years. Patients older than 60 years have an unfavorable prognosis due to resistance to conventional chemotherapy. Volasertib (BI 6727) is a cell-cycle regulator targeting polo-like kinase which has been evaluated in clinical trials in AML. We evaluated effects of volasertib in primary patient samples and NK cells. At equivalent doses, volasertib is cytotoxic to AML blasts but largely spares healthy NK cells. We then evaluated the effect of volasertib treatment in combination with BI 836858 on primary AML blast samples using antibody-dependent cellular cytotoxicity (ADCC) assays. Volasertib treatment of NK cells did not impair NK function as evidenced by comparable levels of BI 836858 mediated ADCC in both volasertib-treated and control-treated NK cells. In summary, volasertib is cytotoxic to AML blasts while sparing NK cell viability and function. Higher BI 836858 mediated ADCC was observed in patient samples pretreated with volasertib. These findings provide a strong rationale to test combination of BI 836858 and volasertib in AML.

Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation.

BACKGROUND AND PURPOSE: Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC). EXPERIMENTAL APPROACH: BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured. KEY RESULTS: Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹79 via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O2⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO. CONCLUSION AND IMPLICATIONS: Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases.

Synthesis and electrochemical and biological studies of novel coumarin-chalcone hybrid compounds.

A series of novel hydroxy-coumarin-chalcone hybrid compounds 2a-i has been synthesized by employing a simple and efficient methodology. An electrochemical characterization using cyclic voltammetry and ESR spectroscopy were carried out to characterize the oxidation mechanism for the target compounds. The antioxidant capacity and reactivity were determined by ORAC and ESR assays, respectively. Biological assays were assessed to evaluate the cytotoxicity and cytoprotection capacity against ROS/RNS on BAEC. The results revealed that all tested compounds present ORAC values that are much higher than other well-known antioxidant compounds such as quercetin and catechin. Compound 2e showed the highest ORAC value (14.1) and also presented a low oxidation potential, good scavenging capacity against hydroxyl radicals, low cytotoxicity, and high cytoprotective activity.

Detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation, and ultimately to oxidative stress leading to cell injury or death. Superoxide radical anion (O2•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2•- in a process called uncoupling, which is believed to be caused by oxidation of heme or the co-factor, tetrahydrobiopterin (BH4). There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production. The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO, the phosphoryl-substituted DEPMPO, and the ester-substituted, EMPO and BMPO, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct.

Inhibition of ROS-induced apoptosis in endothelial cells by nitrone spin traps via induction of phase II enzymes and suppression of mitochondria-dependent pro-apoptotic signaling.

Oxidative stress is the main etiological factor behind the pathogenesis of various diseases including inflammation, cancer, cardiovascular and neurodegenerative disorders. Due to the spin trapping abilities and various pharmacological properties of nitrones, their application as therapeutic agent has been gaining attention. Though the antioxidant properties of the nitrones are well known, the mechanism by which they modulate the cellular defense machinery against oxidative stress is not well investigated and requires further elucidation. Here, we have investigated the mechanisms of cytoprotection of the nitrone spin traps against oxidative stress in bovine aortic endothelial cells (BAEC). Cytoprotective properties of both the cyclic nitrone 5,5-dimethyl-pyrroline N-oxide (DMPO) and linear nitrone α-phenyl N-tert-butyl nitrone (PBN) against H2O2-induced cytotoxicity were investigated. Preincubation of BAEC with PBN or DMPO resulted in the inhibition of H2O2-mediated cytotoxicity and apoptosis. Nitrone-treatment resulted in the induction and restoration of phase II antioxidant enzymes via nuclear translocation of NF-E2-related factor 2 (Nrf-2) in oxidatively-challenged cells. Furthermore, the nitrones were found to inhibit the mitochondrial depolarization and subsequent activation of caspase-3 induced by H2O2. Significant down-regulation of the pro-apoptotic proteins p53 and Bax, and up-regulation of the anti-apoptotic proteins Bcl-2 and p-Bad were observed when the cells were preincubated with the nitrones prior to H2O2-treatment. It was also observed that Nrf-2 silencing completely abolished the protective effects of nitrones. Hence, these findings suggest that nitrones confer protection to the endothelial cells against oxidative stress by modulating phase II antioxidant enzymes and subsequently inhibiting mitochondria-dependent apoptotic cascade.