Nianyu Li

DPI induces mitochondrial superoxide-mediated apoptosis

The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.

Investigations of phagosomes, mitochondria, and acidic granules in human neutrophils using fluorescent probes

The oxidative burst is frequently evaluated by the conversion of dihydrorhodamine 123 (DHR) to rhodamine 123 (R123) and hydroethidium (HE) to ethidium with the use of flow cytometry (FCM). Added R123 accumulates in mitochondria, but during phagocytosis R123 originating from DHR has been observed in neutrophil granules. The present study was designed to identify the site of reactive oxygen species (ROS) formation and the intracellular traffic of R123 in neutrophils by using mitochondrial membrane potential probes and the lysosomotropic probe LysoTracker Red, which have not previously been applied to neutrophils. Quiescent and phagocytosing human peripheral blood neutrophils were incubated with DHR, HE, R123, MitoTracker Green (MTG), MitoTracker Red (CMX‐Ros), and LysoTracker Red alone and in all combinations of red and green probes, and studied by FCM and confocal laser scanning microscopy (CLSM). Phagosomes were filled with R123 originating from DHR. Phagocytosis also triggered the oxidative burst in oxidative response granules that differed from acidic granules. All the neutrophils stained with mitochondrial and lysosomotropic dyes. Added R123 and MTG selectively accumulated in mitochondria. Added R123, MTG, and DHR increased the fluorescence of CMX‐Ros and LysoTracker Red. This is the first FCM and CLSM demonstration of ROS formation in phagosomes. A distinct subpopulation of neutrophil granules, termed oxidative response granules, also was identified. Neutrophil mitochondrial membrane potential may be evaluated by incubating the cells with R123 and MTG, but results with CMX‐Ros should be interpreted with caution. HE and DHR seem to measure a common pathway in the oxidative burst. The simultaneous application of several probes for investigations of organelles carries the risk of probe interference. Cytometry Part B (Clin. Cytometry) 51B:21–29, 2003.

Detection of reactive oxygen species by flow cytometry after spinal cord injury

The monitoring of reactive oxygen species (ROS) levels in injured nervous tissue is critical for both studying the mechanism of secondary damage and evaluating the effectiveness of antioxidants. Flow cytometry is an excellent method to detect ROS in cultured cells and naturally suspended individual cells. However, its use in nervous tissue is limited due to the difficulties in obtaining single cells in suspension. We have developed a new method which minimizes the error during conventional dissociation. Specifically, we introduced a fixation step (with formaldehyde) between the dye loading and dissociation. As a result, the post-injury ROS signals detected by flow cytometry increase significantly when using hydroethidine as superoxide indicator. The injury-induced elevation of ROS obtained from this new method was also in better agreement with the two other standard ROS detection methods, fluorescence microscopy and lipid peroxidation assay. Furthermore, more pronounced decrease of ROS was found in this improved method in response to treatment with a superoxide scavenger, manganese(III)tetrakis(4-benzoic acid)porphyrin. Based on these observations, we suggest that the data obtained from the cells by this new method are more accurate than those from the classic cell dissociation method that dissociates cells directly from fresh tissues.

Differential Mitochondrial Toxicity Screening and Multi- Parametric Data Analysis

Early evaluation of new drug entities for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. Multi-parametric high-content screening (mp-HCS) of mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In this study, we have developed a mp-HCS and multi-parametric data analysis scheme for assessing cell responses to induced mitochondrial perturbation. The mp-HCS measurements are shown to be robust enough to allow for quantitative comparison of biological systems with different metabolic pathways simulated by alteration of growth media. Substitution of medium glucose for galactose sensitized cells to drug action and revealed novel response parameters. Each compound was quantitatively characterized according to induced phenotypic changes of cell morphology and functionality measured by fluorescent biomarkers for mitochondrial activity, plasma membrane permeability, and nuclear morphology. Descriptors of drug effects were established by generation of a SCRIT (Specialized-Cell-Response-to-Induced-Toxicity) vector, consisting of normalized statistical measures of each parameter at each dose and growth condition. The dimensionality of SCRIT vectors depends on the number of parameters chosen, which in turn depends on the hypothesis being tested. Specifically, incorporation of three parameters of response into SCRIT vectors enabled clustering of 84 training compounds with known pharmacological and toxicological activities according to the degree of toxicity and mitochondrial involvement. Inclusion of 6 parameters enabled the resolution of more subtle differences between compounds within a common therapeutic class; scoring enabled a ranking of statins in direct agreement with clinical outcomes. Comparison of drug-induced changes required variations in glucose for separation of mitochondrial dysfunction from other types of cytotoxicity. These results also demonstrate that the number of drugs in a training set, the choice of parameters used in analysis, and statistical measures are fundamental for specific hypothesis testing and assessment of quantitative phenotypic differences.

The increase of reactive oxygen species and their inhibition in an isolated guinea pig spinal cord compression model

Study design: In vitro studies using isolated guinea pig spinal cord.Objectives: To develop an alternative model using isolated guinea pig spinal cord, which can be used to screen antioxidants for in vivo SCI treatment.Setting: Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA.Methods The compression injury was induced by a constant-displacement of 5-s compression of spinal cord using a modified forceps possessing a spacer. Reactive oxygen species (ROS) were evaluated using three distinct methods: fluorescence microscopy, lipid peroxidation assay, and flow cytometry.Results: The injury-mediated ROS increases are comparable with other in vivo studies and consistent with our previous observation using a similar injury model and measured with electrophysiological and anatomical technique. Further, ascorbic acid, hypothermia, or the combination of both significantly suppressed superoxide and lipid peroxidation. The combination treatment was the most effective when compared with ascorbic acid or hypothermia alone.Conclusion: This in vitro model has the advantage of replicating some of the in vivo conditions while gaining the ability to control the experimental conditions. This in vitro model is suitable to study the mechanisms of ROS generation and degradation and can also be used to critically evaluate the effective suppressor of ROS in the contents of spinal cord traumatic injury.

Homeostasis of Human NK Cells Is Not IL-15 Dependent

IL-15 is a proinflammatory cytokine that plays an important role in the development and activation of NK cells and is a potential target for inflammatory disease therapy. Studies conducted in IL-15- and IL-15R knockout mice identified IL-15 as an important cytokine for NK cell homeostasis. Consistent with this information derived from genetically modified mice, we demonstrated that neutralizing IL-15 with a mouse anti-mouse IL-15 mAb (M96) depletes C57BL/6 mouse NK cells. An mAb directed against macaque IL-15 (Hu714MuXHu) was manufactured and demonstrated to block IL-15–induced activation of nonhuman primate (NHP) NK cells in vitro. Neutralization of macaque IL-15 by parenteral administration of Hu714MuXHu reduces (>95%) circulating NK cell counts in NHPs. A blocking mAb directed against human IL-15 (huIL-15; AMG 714) was manufactured. Unexpectedly, when human subjects were treated with the blocking anti–IL-15 Ab AMG 714 in clinical trials, no reductions in circulating NK cell counts were observed despite achieving significantly higher exposures than the levels of Hu714MuXHu needed to cause NK cell count reductions in NHPs in vivo. Both AMG 714 and Hu714MuXHu are able to block huIL-15 activity in a human T cell blast proliferation and IFN-γ production assay. Both Abs block huIL-15–mediated Stat5 activation and CD69 expression in human NK cells. Collectively, these results demonstrate that NK cell homeostasis is obligatorily dependent upon IL-15 in both mice and NHPs, but that IL-15 is dispensable for maintenance of circulating human NK cells.

Unexpected thrombocytopenia and anemia in cynomolgus monkeys induced by a therapeutic human monoclonal antibody.

Cynomolgus monkeys dosed with a therapeutic monoclonal antibody (mAbY.1) at ≥50 mg/kg had unexpected acute thrombocytopenia (nadir ∼3,000 platelets/µl), sometimes with decreases in red cell mass. Increased activated macrophages, mitotic figures, and erythrophagocytosis were observed in the spleen. Binding of mAbY.1 to cynomolgus peripheral blood cells could not be detected in vitro. mAbY.1 induced phagocytosis of platelets by peripheral blood monocytes from cynomolgus monkeys, but not from humans. mAbs sharing the same constant domain (Fc) sequences, but differing from mAbY.1 in their variable domains, bound competitively to and had similar biological activity against the intended target. None of these antibodies had hematologic liabilities in vitro or in vivo. Neither the F(ab’)2 portion of mAbY.1 nor the F(ab’)2 portion on an aglycosylated Fc (IgG1) framework caused phagocytosis of platelets in vitro. These data suggest that the hematologic effects of mAbY.1 in cynomolgus monkeys likely occurred through an off-target mechanism, shown to be driven by 1 to 3 amino acid differences in the light chain. The hematologic effects made mAbY.1 an unsuitable candidate for further development as a therapeutic agent. This example demonstrates that nonclinical safety studies may be essential for understanding off-target effects of mAbs prior to clinical trials.

High-Throughput Secondary Screening at the Single-Cell Level

We have developed an automated system for drug screening using a single-cell–multiple functional response technology. The approach uses a semiautomated preparatory system, high-speed sample collection, and a unique analytical tool that provides instantaneous results for compound dilutions using 384-well plates. The combination of automation and rapid robotic sampling increases quality control and robustness. High-speed flow cytometry is used to collect single-cell results together with a newly defined analytical tool for extraction of IC50 curves for multiple assays per cell. The principal advantage is the extreme speed of sample collection, with results from a 384-well plate being completed for both collection and data processing in less than 10 min. Using this approach, it is possible to extract detailed drug response information in a highly controlled fashion. The data are based on single-cell results, not populations. With simultaneous assays for different functions, it is possible to gain a more detailed understanding of each drug/compound interaction. Combined with integrated advanced data processing directly from raw data files, the process from sampling to analytical results is highly intuitive. Direct PubMed links allow review of drug structure and comparisons with similar compounds.

A Systematic Assessment Of Mitochondrial Function Identified Novel Signatures For Drug-Induced Mitochondrial Disruption In Cells

Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability.

Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro.

The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem. 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro. These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions.