Interactions of binary mixtures of metals on rainbow trout (Oncorhynchus mykiss) heart mitochondrial H2O2 homeodynamics.

For continuous pumping of blood, the heart needs a constant supply of energy (ATP) that is primarily met via oxidative phosphorylation in the mitochondria of cardiomyocytes. However, sustained high rates of electron transport for energy conversion redox reactions predisposes the heart to the production of reactive oxygen species (ROS) and oxidative stress. Mitochondrial ROS are fundamental drivers of responses to environmental stressors including metals but knowledge of how combinations of metals alter mitochondrial ROS homeodynamics remains sparse. We explored the effects and interactions of binary mixtures of copper (Cu), cadmium (Cd), and zinc (Zn), metals that are common contaminants of aquatic systems, on ROS (hydrogen peroxide, H2O2) homeodynamics in rainbow trout (Oncorhynchus mykiss) heart mitochondria. Isolated mitochondria were energized with glutamate-malate or succinate and exposed to a range of concentrations of the metals singly and in equimolar binary concentrations. Speciation analysis revealed that Cu was highly complexed by glutamate or Tris resulting in Cu2+ concentrations in the picomolar to nanomolar range. The concentration of Cd2+ was 7.2-7.5 % of the total while Zn2+ was 15 % and 21 % of the total during glutamate-malate and succinate oxidation, respectively. The concentration-effect relationships for Cu and Cd on mitochondrial H2O2 emission depended on the substrate while those for Zn were similar during glutamate-malate and succinate oxidation. Cu + Zn and Cu + Cd mixtures exhibited antagonistic interactions wherein Cu reduced the effects of both Cd and Zn, suggesting that Cu can mitigate oxidative distress caused by Cd or Zn. Binary combinations of the metals acted additively to reduce the rate constant and increase the half-life of H2O2 consumption while concomitantly suppressing thioredoxin reductase and stimulating glutathione peroxidase activities. Collectively, our study indicates that binary mixtures of Cu, Zn, and Cd act additively or antagonistically to modulate H2O2 homeodynamics in heart mitochondria.

A model naphthenic acid decouples oxidative phosphorylation through selective inhibition of mitochondrial complex activity.

The naphthenic acid fraction compound (NAFC), 3,5-dimethyladamantane-1-acetic acid, was tested for its ability to uncouple mitochondrial oxidative phosphorylation. Mitochondria isolated from rainbow trout (Oncorhynchus mykiss) liver were exposed to 3,5-dimethyladamantane-1-acetic acid in state 3 and 4 respiration, and mitochondrial membrane potential were quantified. Electron transport chain (ETC) protein complexes were isolated using pharmacological agents and inhibitors, and their activities measured. The NAFC compound completely inhibited states 3 and 4 respiration with an IC50 of 0.77 and 1.01 mM, respectively. The NAFC compound partially uncoupled mitochondrial membrane potential in state 3 and 4 respiration with an IC50 of 2.19 and 1.73 mM, respectively. The NAFC impaired the activities of ETC protein complexes with a 9.5-fold range in sensitivity. The relative inhibitory effect of the ETC protein complexes to NAFC was CIV≥CI>CIII>CII. The impairment of mitochondrial oxidative phosphorylation by adamantane 3,5-dimethyladamantane-1-acetic acid is mediated via its inhibition of ETC protein complexes.

A phase 1/2 clinical trial of invariant natural killer T cell therapy in moderate-severe acute respiratory distress syndrome.

Invariant natural killer T (iNKT) cells, a unique T cell population, lend themselves for use as adoptive therapy due to diverse roles in orchestrating immune responses. Originally developed for use in cancer, agenT-797 is a donor-unrestricted allogeneic ex vivo expanded iNKT cell therapy. We conducted an open-label study in virally induced acute respiratory distress syndrome (ARDS) caused by the severe acute respiratory syndrome-2 virus (trial registration NCT04582201). Here we show that agenT-797 rescues exhausted T cells and rapidly activates both innate and adaptive immunity. In 21 ventilated patients including 5 individuals receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO), there are no dose-limiting toxicities. We observe an anti-inflammatory systemic cytokine response and infused iNKT cells are persistent during follow-up, inducing only transient donor-specific antibodies. Clinical signals of associated survival and prevention of secondary infections are evident. Cellular therapy using off-the-shelf iNKT cells is safe, can be rapidly scaled and is associated with an anti-inflammatory response. The safety and therapeutic potential of iNKT cells across diseases including infections and cancer, warrants randomized-controlled trials.

Effect of skeletal muscle mitochondrial phenotype on H2O2 emission.

Reactive oxygen species (ROS) are a key output of the skeletal muscle mitochondrial information processing system both at rest and during exercise. In skeletal muscle, mitochondrial ROS release depends on multiple factors; however, fiber-type specific differences remain ambiguous in part owing to the use of mitochondria from mammalian muscle that consist of mixed fibers. To elucidate fiber-type specific differences, we used mitochondria isolated from rainbow trout (Oncorhynchus mykiss) red and white skeletal muscles that consist of spatially distinct essentially pure red and white fibers. We first characterized the assay conditions for measuring ROS production (as H2O2) in isolated fish red and white skeletal muscle mitochondria (RMM and WMM) and thereafter compared the rates of emission during oxidation of different substrates and the responses to mitochondrial electron transport system (ETS) pharmacological modulators. Our results showed that H2O2 emission rates by RMM and WMM can be quantified using the same protein concentration and composition of the Amplex UltraRed-horseradish peroxidase (AUR-HRP) detection system. For both RMM and WMM, protein normalized H2O2 emission rates were highest at the lowest protein concentration tested and decreased exponentially thereafter. However, the absolute values of H2O2 emission rates depended on the calibration curves used to convert fluorescent signals to H2O2 while the trends depended on the normalization strategy. We found substantial qualitative and quantitative differences between RMM and WMM in the H2O2 emission rates depending on the substrates being oxidized and their concentrations. Similarly, pharmacological modulators of the ETS altered the magnitudes and trends of the H2O2 emission differently in RMM and WMM. While comparable concentrations of substrates elicited maximal albeit quantitively different emission rates in RMM and WMM, different concentrations of pharmacological ETS modulators may be required for maximal H2O2 emission rates depending on muscle fiber-type. Taken together, our study suggests that biochemical differences exist in RMM compared with WMM that alter substrate oxidation and responses to ETS modulators resulting in fiber-type specific mitochondrial H2O2 emission rates.

Fixed-Duration Ibrutinib-Venetoclax in Patients with Chronic Lymphocytic Leukemia and Comorbidities.

BACKGROUND: GLOW is a phase 3 trial evaluating the efficacy and safety of ibrutinib-venetoclax in older patients and/or those with comorbidities with previously untreated chronic lymphocytic leukemia (CLL). METHODS: We randomly assigned (1:1) patients 65 years of age or older or those 18 to 64 years of age who also had a Cumulative Illness Rating Scale (CIRS) score greater than 6 (CIRS scores range from 0 to 56, with higher scores indicating more impaired function of organ systems) or creatinine clearance of less than 70 ml/min, to ibrutinib-venetoclax (3 cycles ibrutinib lead-in, then 12 cycles ibrutinib-venetoclax) or chlorambucil-obinutuzumab (6 cycles). The primary end point was progression-free survival (PFS) assessed by an independent review committee. Secondary end points included undetectable minimal residual disease (uMRD), response rates, and safety. RESULTS: This study enrolled 211 patients, with 106 randomly assigned to ibrutinib-venetoclax and 105 to chlorambucil-obinutuzumab. With a median follow-up of 27.7 months, there were 22 PFS events for ibrutinib-venetoclax and 67 events for chlorambucil-obinutuzumab. PFS was significantly longer for ibrutinib-venetoclax than for chlorambucil-obinutuzumab (hazard ratio, 0.216; 95% confidence interval [CI], 0.131 to 0.357; P<0.001). The improvement in PFS with ibrutinib-venetoclax was consistent across predefined subgroups, including patients 65 years of age or older or with a CIRS score greater than 6. The best uMRD rate in bone marrow by next-generation sequencing was significantly higher for ibrutinib-venetoclax (55.7%) than for chlorambucil-obinutuzumab (21.0%; P<0.001). The proportion of patients with sustained uMRD in peripheral blood from 3 to 12 months after end of treatment was 84.5% for ibrutinib-venetoclax and 29.3% for chlorambucil-obinutuzumab. Four patients treated with ibrutinib-venetoclax required subsequent therapy compared with 27 patients receiving chlorambucil-obinutuzumab (hazard ratio, 0.143; 95% CI, 0.050 to 0.410). Adverse events grade 3 or greater occurred for 80 (75.5%) and 73 (69.5%) patients receiving ibrutinib-venetoclax and chlorambucil-obinutuzumab, respectively, with neutropenia being most common in both arms (37 [34.9%] and 52 [49.5%]). There were 11 (10.4%) and 12 (11.4%) all-cause deaths in the ibrutinib-venetoclax and chlorambucil-obinutuzumab arms, respectively. CONCLUSIONS: Ibrutinib-venetoclax, an all-oral, once-daily, fixed-duration combination, demonstrated superior PFS and deeper and better sustained responses versus chlorambucil-obinutuzumab as first-line CLL treatment in older patients and/or those with comorbidities. (Funded by Janssen Research & Development, LLC, and Pharmacyclics; ClinicalTrials.gov number, NCT03462719.)