Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs.

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk Activity in Mouse Embryonic Stem Cells.

Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.

Adipokinetic hormone-induced antioxidant response in Spodoptera littoralis.

The antioxidative potential of the Manduca sexta adipokinetic hormone (Manse-AKH) in the last instar larvae of Spodoptera littoralis (Noctuidae, Lepidoptera) was demonstrated after exposure to oxidative stress (OS) elicited by feeding on artificial diet containing tannic acid (TA). Determination of protein carbonyls (PCs) and reduced glutathione (GSH) levels, monitoring of activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferases (GSTs), as well as measuring of the mRNA expression of CAT and SOD were used as markers of the OS. Injection of the Manse-AKH (5 pmol per individual) reversed the OS status by mitigation of PCs formation and by stimulation of glutathione-S-transferases (GSTs) activity. The CAT and SOD mRNA expression was significantly suppressed after the Manse-AKH injection while activity of these enzymes was not affected. These results indicate that diminishing of OS after the AKH injection might be a result of activation of specific enzymatic pathway possibly at the post-translational level rather than a direct effect on regulation of antioxidant marker genes at the transcriptional level.

Oxidative stress elicited by insecticides: a role for the adipokinetic hormone.

Adipokinetic hormones (AKHs) are insect neuropeptides responding to stress situations including oxidative stress. Two insecticides - endosulfan and malathion - were used to elicit oxidative stress conditions in the firebug Pyrrhocoris apterus, and the physiological functions of AKHs and their ability to activate protective antioxidative reactions were studied. The insecticide treatments elicited only a slight increase of the AKH level in CNS, but more intensive increase in haemolymph, which indicates an immediate involvement of AKH in the stress response. The treatment also resulted in a significant increase of catalase activity in the bug's body and depletion of the reduced glutathione pool in the haemolymph, however, co-application of the insecticides with the AKH (80 pmol) reduced the effect. It has also been found that co-application of the insecticides with AKH increased significantly the bug mortality compared to that induced by the insecticides alone. This enhanced effect of the insecticides probably resulted from the stimulatory role of AKH on bug metabolism: the carbon dioxide production was increased significantly after the co-treatment by AKH with insecticides compared to insecticide treatment alone. It was hypothesized that the increased metabolic rate could intensify the insecticide action by an accelerated rate of exchange of metabolites accompanied by faster penetration of insecticides into tissues.

20-Hydroxyecdysone prevents oxidative stress damage in adult Pyrrhocoris apterus.

Injections of 38 pmol paraquat (1,1'-dimethyl-4,4'-bypyridilium) into adult Pyrrhocoris apterus (average body weight 29.6 mg in males and 36.9 mg in females) caused a significant elevation of lipid peroxidation and protein carbonylation and a decline of membrane fluidity in the microsomal brain fraction. Another manifestation of oxidative stress was a depletion of the reduced glutathione pool and reduction of the gamma-glutamyl transpeptidase activity in the brain extracts. The damaging action of paraquat on the brain was counteracted by simultaneous injection of 1 pmol 20-hydroxyecdysone (20E). 20E restrained lipid peroxidation and the formation of protein carbonyls, ameliorated changes in microsomal membrane fluidity, enhanced the level of reduced glutathione, and upregulated the activity of gamma-glutamyl transpeptidase. At the organismic level, 20E curtailed three detrimental effects caused by paraquat injection: the disappearance of a blood protein, the suppression of fecundity and egg hatchability, and the shortening of adult life span. The data showed that 20E provided a systemic antioxidant protection but the significance of endogenous ecdysteroids in the management of oxidative stress remains to be shown.

Adipokinetic hormone-induced enhancement of antioxidant capacity of Pyrrhocoris apterus hemolymph in response to oxidative stress.

The in vivo effects of oxidative stress on adipokinetic hormone (AKH) titer in short-winged (brachypterous) males of the firebug Pyrrhocoris apterus were tested using paraquat (PQ), a bipyridilium herbicide. PQ undergoes a cyclic redox reaction with oxygen during microsomal and electron transfer reactions forming free radicals in the insect body. Oxidative insult (40 pmol PQ) resulted in enhanced protein carbonylation (a biomarker for oxidative stress) and a depletion of glutathione (GSH) pool in the hemolymph. Interestingly, AKH titer was significantly enhanced in hemolymph at 4 h post inoculation of PQ, while its content in CNS (brain with corpora cardiaca) showed non-specific changes in comparable period. Co-injection of AKH with PQ (40 pmol each) reversed these effects by decreasing protein carbonyl formation, increasing reduced GSH levels, and enhancing the total antioxidant capacity of cell free plasma. Our results indicate that there is a positive feedback regulation between an oxidative stressor action and the level of AKH in insect body, and that AKHs might be involved in the activation of antioxidant protection mechanism.