A universal dual mechanism immunotherapy for the treatment of influenza virus infections.

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells. We show that this leads to simultaneous inhibition of virus release, and immune-mediated elimination of both free virus and virus-infected cells. Intranasal or intraperitoneal administration of a single dose of drug to mice infected with 100x MLD50 virus is shown to eradicate advanced infections from representative strains of both influenza A and B viruses. Since treatments of severe infections remain effective up to three days post lethal inoculation, our approach may successfully treat infections refractory to current therapies.

Prenatal microRNA miR-200b Therapy Improves Nitrofen-induced Pulmonary Hypoplasia Associated With Congenital Diaphragmatic Hernia.

OBJECTIVE: We aimed to evaluate the use of miR-200b as a prenatal transplacental therapy in the nitrofen rat model of abnormal lung development and congenital diaphragmatic hernia (CDH). BACKGROUND: Pulmonary hypoplasia (PH) and pulmonary hypertension determine mortality and morbidity in CDH babies. There is no safe medical prenatal treatment available. We previously discovered that higher miR-200b is associated with better survival in CDH babies. Here, we investigate the role of miR-200b in the nitrofen rat model of PH and CDH and evaluate its use as an in vivo prenatal therapy. METHODS: We profiled miR-200b expression during nitrofen-induced PH using RT-qPCR and in situ hybridization in the nitrofen rat model of PH and CDH. The effects of nitrofen on downstream miR-200b targets were studied in bronchial lung epithelial cells using a SMAD luciferase assay, Western blotting and Immunohistochemistry. We evaluated miR-200b as a lung growth promoting therapy ex vivo and in vivo using lung explant culture and transplacental prenatal therapy in the nitrofen rat model. RESULTS: We show that late lung hypoplasia in CDH is associated with (compensatory) upregulation of miR-200b in less hypoplastic lungs. Increasing miR-200b abundance with mimics early after nitrofen treatment decreases SMAD-driven TGF-ß signaling and rescues lung hypoplasia both in vitro and in vivo. Also, prenatal miR-200b therapy decreases the observed incidence of CDH. CONCLUSIONS: Our data indicate that miR-200b improves PH and decreases the incidence of CDH. Future studies will further exploit this newly discovered prenatal therapy for lung hypoplasia and CDH.

Does bird metabolic rate influence mosquito feeding preference?

BACKGROUND: Host selection by mosquitoes plays a central role in the transmission of vector-borne infectious diseases. Although interspecific variation in mosquito attraction has often been reported, the mechanisms underlying intraspecific differences in hosts' attractiveness to mosquitoes are still poorly known. Metabolic rate is related to several physiological parameters used as location cues by mosquitoes, and so potentially affect host-vector contact rates. Therefore, individual hosts with higher metabolic rates should be more attractive to host-seeking mosquitoes. Here, we experimentally investigated the role of bird metabolic rate in the feeding preferences of Culex pipiens (Linnaeus), a widespread mosquito vector of many pathogens affecting human and wildlife health. RESULTS: Passer domesticus (Linnaeus) pairs containing one bird treated with 2,4-dinitrophenol (DNP) and the other injected with phosphate-buffered saline solution (PBS) (i.e. control) were simultaneously exposed overnight to mosquitoes. The treatment did not affect the proportion of mosquitoes biting on each individual. However, mosquito feeding preference was negatively associated with bird resting metabolic rate but positively with bird body mass. These two variables explained up to 62.76% of the variations in mosquito feeding preference. CONCLUSIONS: The relationships between mosquito feeding preferences and individual host characteristics could be explained by enhanced anti-mosquito behaviour associated with higher metabolic rates. The potential role of cues emitted by hosts is also discussed. Thus, individuals with high metabolism may actively avoid being bitten by mosquitoes, despite releasing more attractant cues. Since metabolic rates can be related to individual differences in personality and life history traits, differences in mosquitoes' feeding preferences may be related to intraspecific differences in exposure to vector-borne pathogens.

Being in control? A thematic content analysis of 14 in-depth interviews with 2,4-dinitrophenol users.

BACKGROUND: 2,4-Dinitrophenol (2,4-DNP) is a compound with multiple industrial purposes. Currently unlicensed for human consumption, it is used by the gym-going population for drastic, short-term body fat loss. Nonetheless, physiological mechanisms can lead to potentially fatal hyperthermia. Reported fatal incidents have caused concern and highlighted the need for intervention. Understanding decision-making leading to 2,4-DNP use alongside the perceived outgroup attitudes is vital to forming effective harm minimisation policies targeting current and potential users. First-hand accounts from this elusive population are scarce. METHODS: Fourteen novel and experienced users (13 male, 1 female) were recruited via "snowballing" techniques. Semi-structured interviews were conducted, comprising 28 questions. Thematic content analysis was conducted using 37 codes. RESULTS: Four characteristic themes emerged: 1. Users considered the Internet to be a crucial multifunctional resource directly impacting their 2,4-DNP use. 2. Users "respected" 2,4-DNP, proactively taking harm reduction measures. 3. Attitudinal polarisation towards 2,4-DNP within the gym-going community was consistent in all accounts. 4. Users perceived outgroup populations to have inherently negative attitudes towards their use. These themes fell under the all-encompassing theme of "being in control". CONCLUSION: For the first time, this study offers a rich detail of attitudes toward 2,4-DNP use by giving a collective voice to users. The element of control over every aspect of the users' life appears to be a significant contributor to the successful risk-management of 2,4-DNP use. In the absence of an established safe upper limit and effective regulatory control, education is critical to harm minimisation.

Sustained-release mitochondrial protonophore reverses nonalcoholic fatty liver disease in rats.

As a mitochondrial uncoupler, 2,4-dinitrophenol (DNP) is proven therapeutically effective against nonalcoholic fatty liver disease (NAFLD) by uncoupling oxidation and phosphorylation. However, a major factor that impedes the clinical application of DNP is the significant side effects derived from its frequent hyperthermia and even death. In this study, we developed an injectable liquid crystal gel (DNP-LC-gel) to reduce the toxicity of DNP. DNP-LC-gel achieved sustained release and maintained DNP plasma concentration at an effective drug level. In a rat model of NAFLD, DNP-LC-gel treated rats reduced hepatic steatosis, liver triglyceride content, plasma triacylglycerol (TG) and total cholesterol (TC) content. Compared with DNP solution (DNP-soln), rats after DNP-LC-gel treatment showed no body temperature toxicity and local irritation. All results above indicated that DNP-LC-gel has a great potential for NAFLD therapy.

DNP, mitochondrial uncoupling, and neuroprotection: A little dab'll do ya.

Recent findings have elucidated roles for mitochondrial uncoupling proteins (UCPs) in neuronal plasticity and resistance to metabolic and oxidative stress. UCPs are induced by bioenergetic challenges such as caloric restriction and exercise and may protect neurons against dysfunction and degeneration. The pharmacological uncoupler 2,4-dinitrophenol (DNP), which was once prescribed to >100,000 people as a treatment for obesity, stimulates several adaptive cellular stress-response signaling pathways in neurons including those involving the brain-derived neurotrophic factor (BDNF), the transcription factor cyclic AMP response element-binding protein (CREB), and autophagy. Preclinical data show that low doses of DNP can protect neurons and improve functional outcome in animal models of Alzheimer's and Parkinson's diseases, epilepsy, and cerebral ischemic stroke. Repurposing of DNP and the development of novel uncoupling agents with hormetic mechanisms of action provide opportunities for new breakthrough therapeutic interventions in a range of acute and chronic insidious neurodegenerative/neuromuscular conditions, all paradoxically at body weight-preserving doses.

Preconditioning of mesenchymal stem cells with 2,4-dinitrophenol improves cardiac function in infarcted rats.

AIMS: The aim of this study is to determine if preconditioning of bone marrow derived mesenchymal stem cells (MSCs) with 2,4-dinitrophenol (DNP) improves survival of transplanted stem cells in a rat model of myocardial infarction (MI), and to asses if this strategy has measurable impact on cardiac function. MAIN METHODS: MSCs were preconditioned with DNP. In vitro cell adhesion assay and qRT-PCR were performed to analyze the expression of genes involved in cardiomyogenesis, cell adhesion and angiogenesis. MI was produced by occlusion of left anterior descending coronary artery. One million cells were transplanted by intramyocardial injection into the infarcted myocardium. Echocardiography was performed after two and four weeks of cellular transplantation. Hearts were harvested after four weeks and processed for histological analysis. KEY FINDINGS: DNP treated MSCs adhered to the surface more (p<0.001) as compared to the normal MSCs. Gene expression levels were significantly upregulated in case of DNP treatment. The number of viable MSCs was more (p<0.001) in animals that received DNP treated MSCs, leading to significant improvement in cardiac function. Histological analysis revealed significant reduction in scar formation (p<0.001), maintenance of left ventricular wall thickness (p<0.001), and increased angiogenesis (p<0.01). SIGNIFICANCE: The study evidenced for the first time that MSCs preconditioned with DNP improved cardiac function after transplantation. This can be attributed to improved survival, homing, adhesion, and cardiomyogenic and angiogenic differentiation of DNP treated MSCs in vivo.

Persistent reduced oxygen requirement following blood transfusion during recovery from hemorrhagic shock.

Our study intended to determine the effects on oxygen uptake (VO2) of restoring a normal rate of O2 delivery following blood transfusion (BT) after a severe hemorrhage (H). Spontaneously breathing urethane anesthetized rats were bled by removing 20 ml/kg of blood over 30 min. Rats were then infused with their own shed blood 15 min after the end of H. At mid-perfusion, half of the rats received a unique infusion of the decoupling agent 2,4-dinitrophenol (DNP, 6 mg/kg). VO2 and arterial blood pressure (ABP) were continuously measured throughout the study, along with serial determination of blood lactate concentration [La]. Animals were euthanized 45 min after the end of reperfusion; liver and lungs were further analyzed for early expression of oxidative stress gene using RT-PCR. Our bleeding protocol induced a significant decrease in ABP and increase in [La], while VO2 dropped by half. The O2 deficit progressively accumulated during the period of bleeding reached -114 ± 53 ml/kg, just before blood transfusion. Despite the transfusion of blood, a significant O2 deficit persisted (-82 ± 59 ml/kg) 45 min after reperfusion. This slow recovery of VO2 was sped up by DNP injection, leading to a fast recovery of O2 deficit after reperfusion, becoming positive (+460 ± 132 ml/kg) by the end of the protocol, supporting the view that O2 supply is not the main controller of VO2 dynamics after BT. Of note is that DNP also enhanced oxidative stress gene expression (up-regulation of NADPH oxidase 4 in the lung for instance). The mechanism of slow recovery of O2 requirement/demand following BT and the resulting effects on tissues exposed to relatively high O2 partial pressure are discussed.

Controlled-release mitochondrial protonophore reverses diabetes and steatohepatitis in rats.

Nonalcoholic fatty liver disease (NAFLD) is a major factor in the pathogenesis of type 2 diabetes (T2D) and nonalcoholic steatohepatitis (NASH). The mitochondrial protonophore 2,4 dinitrophenol (DNP) has beneficial effects on NAFLD, insulin resistance, and obesity in preclinical models but is too toxic for clinical use. We developed a controlled-release oral formulation of DNP, called CRMP (controlled-release mitochondrial protonophore), that produces mild hepatic mitochondrial uncoupling. In rat models, CRMP reduced hypertriglyceridemia, insulin resistance, hepatic steatosis, and diabetes. It also normalized plasma transaminase concentrations, ameliorated liver fibrosis, and improved hepatic protein synthetic function in a methionine/choline-deficient rat model of NASH. Chronic treatment with CRMP was not associated with any systemic toxicity. These data offer proof of concept that mild hepatic mitochondrial uncoupling may be a safe and effective therapy for the related epidemics of metabolic syndrome, T2D, and NASH.

Chronic mitochondrial uncoupling treatment prevents acute cold-induced oxidative stress in birds.

Endotherms have evolved two major types of thermogenesis that allow them to actively produce heat in response to cold exposure, either through muscular activity (i.e. shivering thermogenesis) or through futile electro-chemical cycles (i.e. non-shivering thermogenesis). Amongst the latter, mitochondrial uncoupling is of key importance because it is suggested to drive heat production at a low cost in terms of oxidative stress. While this has been experimentally shown in mammals, the oxidative stress consequences of cold exposure and mitochondrial uncoupling are clearly less understood in the other class of endotherms, the birds. We compared metabolic and oxidative stress responses of zebra finches chronically treated with or without a chemical mitochondrial uncoupler (2,4-dinitrophenol: DNP), undergoing an acute (24 h) and a chronic (4 weeks) cold exposure (12 °C). We predicted that control birds should present at least a transient elevation of oxidative stress levels in response to cold exposure. This oxidative stress cost should be more pronounced in control birds than in DNP-treated birds, due to their lower basal uncoupling state. Despite similar increase in metabolism, control birds presented elevated levels of DNA oxidative damage in response to acute (but not chronic) cold exposure, while DNP-treated birds did not. Plasma antioxidant capacity decreased overall in response to chronic cold exposure. These results show that acute cold exposure increases oxidative stress in birds. However, uncoupling mitochondrial functioning appears as a putative compensatory mechanism preventing cold-induced oxidative stress. This result confirms previous observations in mice and underlines non-shivering thermogenesis as a putative key mechanism for endotherms in mounting a response to cold at a low oxidative cost.

2,4-dinitrophenol partially alleviates ferrocyanide-induced toxicity in Drosophila melanogaster.

The toxicity of potassium ferrocyanide (PFC) and protective effects of 2,4-dinitrophenol (DNP) under PFC treatment were tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with PFC at concentrations of 1.0 mM and mixtures with DNP in concentrations of 0.50 and 1.25 mM, either alone or in combination with 1.0 mM PFC. Food supplementation with PFC decreased larvae viability or pupation height, whereas when larvae were fed by PFC and DNP combination the decrease was less pronounced. Larval exposure to PFC and mixtures of DNP and PFC lowered activities of aconitase. Larval treatment with PFC resulted in higher carbonyl protein, uric acid, and low molecular mass thiols content and higher activity of thioredoxin reductase in adult flies, while DNP in mixtures with PFC relieved these effects. Furthermore, treatment with PFC/DNP mixtures resulted in higher activities of superoxide dismutase and glutathione-S-transferase. It is proposed that PFC toxicity is mainly related to the cyanide and iron ions, released during its decomposition. The potential mechanisms of protective DNP effects against PFC toxicity are discussed.

The mitochondrial uncoupler 2,4-dinitrophenol attenuates sodium nitroprusside-induced toxicity in Drosophila melanogaster: potential involvement of free radicals.

The toxicity of sodium nitroprusside (SNP) (an inducer of oxidative/nitrosative stress) and the attenuation of SNP effects by 2,4-dinitrophenol (DNP) (that induces mild uncoupling of respiration) were evaluated in the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with 1.0 mM SNP, 0.5 or 1.25 mM DNP, or with mixtures 1.0 mM SNP plus 0.5 or 1.25 mM DNP. Food supplementation with SNP decreased larval viability and pupation height whereas supplementation with DNP substantially reversed these changes. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control larvae and larvae fed on food supplemented with SNP, DNP, or SNP/DNP mixtures. Larval exposure to SNP lowered activities of aconitase, while the presence of DNP reduced the negative impact of SNP by raising aconitase activity back to near control levels. Larval treatment with SNP also elevated the contents of carbonyl protein, uric acid and low molecular mass thiols and produced higher activities of superoxide dismutase, glutathione S-transferase, glucose-6-phosphate dehydrogenase and thioredoxin reductase in adult flies. However, the presence of DNP in the food mixtures prevented SNP-induced changes in thioredoxin reductase and glucose-6-phosphate dehydrogenase activities, as well as uric acid and low-molecular-mass thiol content. The potential mechanisms by which DNP exerts protective effects against SNP toxicity are discussed.

2, 4-dinitrophenol poisoning caused by non-oral exposure.

2, 4-Dinitrophenol (2, 4-DNP) is widely used in industry, but recently, poisoning through consumption for weight control has been frequently reported. We report the cases of two patients whose deaths were attributed to occupational and non-oral exposure of 2, 4-DNP. They were all poisoned through skin absorption and respiratory tract inhalation; common features were excessive sweating, hyperthermia, tachycardia, clouded consciousness and asystole. Because of the lack of specific early symptoms, effective antidotes and the means of washing the contamination from the skin, their arrival in hospital was delayed and the supportive therapy was ineffectual. Cardiac arrest occurred quickly and unexpected after admission.

Effects of 2,4-dinitrophenol on ischemia-induced blood-brain barrier disruption.

This study examines the effect of 2,4-dinitrophenol (DNP), a mitochondrial uncoupling agent, during focal brain ischemia induced by middle cerebral artery (MCA) occlusion. Blood-brain barrier (BBB) disruption was assessed after 2 hours of occlusion with 2 hours of reperfusion or 4 hours of permanent occlusion by measurement of the influx rate constant (K(i)) for 3H-inulin in the MCA territory ipsi- and contralateral to the occlusion. Three experimental groups were examined: vehicle and 1 and 5 mg/kg DNP treated animals (given 30 minutes prior to occlusion). Four hours of permanent MCA occlusion only induced a modest increase in the K(i) for inulin in vehicle-treated animals (0.09 +/- 0.01 vs. 0.07 +/- 0.01 microL/g/min in contralateral tissue). Although 5 mg/kg DNP significantly increased this disruption (p < 0.01), this effect was relatively minor (0.14 +/- 0.02 microL/g/min). In contrast, DNP treatment in transient ischemia markedly increased barrier disruption. The ipsilateral K(i) for 3H-inulin were 0.15 +/- 0.04, 0.37 +/- 0.06, and 0.79 +/- 0.17 microL/g/min in vehicle, 1 mg/kg DNP and 5 mg/kg DNP groups, respectively. DNP did not induce barrier disruption in the contralateral hemisphere. Thus, while there is evidence that DNP can be neuroprotective, it has adverse effects on the BBB during ischemia, particularly with reperfusion. Considering the importance of naturally- or therapeutically-induced reperfusion in limiting brain damage, this may limit the utility of DNP and mitochondrial uncouplers as therapeutic agents.

Entacapone does not induce conformational changes in liver mitochondria or skeletal muscle in vivo.

Entacapone and tolcapone, novel catechol-O-methyl-transferase (COMT) inhibitors, have been developed for the treatment of Parkinson's disease in combination with levodopa. Three fatal cases of drug-induced hepatitis, one with hepatic necrosis and mitochondrial changes have been reported in clinical use of tolcapone. In vitro tolcapone has been shown to induce uncoupling of oxidative phosphorylation. Liver and skeletal muscle tissues from an oral rat toxicity study were used to investigate the influence of entacapone, tolcapone (300 and 500 mg/kg/day) or a known uncoupling agent, 2,4-dinitrophenol (DNP), (20 mg/kg/ day) on the cell morphology. Centrolobular hypertrophy was revealed in the histopathology of the liver in tolcapone-treated rats. Transmission electron microscopy (TEM) of the liver and skeletal muscle tissue, revealed mitochondrial swelling and reduced matrix density with deformation of cristae in the tolcapone and DNP groups. Intermyofibrillar edema was characteristic of the skeletal muscle tissue of DNP- and tolcapone-exposed animals. In the tolcapone group, also the sarcomeres were prominent. Treatment-related light microscopic or TEM findings were not observed either in entacapone-treated or control animals. The similarity of structural damages induced by both tolcapone- and DNP suggests that uncoupling of oxidative phosphorylation may contribute to the toxicity of tolcapone in the rat.

Defective endogenous nitric oxide-mediated modulation of cellular respiration in canine skeletal muscle after the development of heart failure.

BACKGROUND: It is well documented that nitric oxide (NO) suppresses the function of a number of mitochondrial enzymes. Our recent studies found that endogenous NO may play an important role in the modulation of tissue oxygen (O2) consumption and cellular respiration both in vitro and in vivo. METHODS: Tissue O2 consumption was measured by a Clark-type O2 electrode at 37 degrees C in freshly isolated skeletal muscle segments from the accessory head of the triceps brachii (90% type I muscle fiber) and extensor carpi radialis (86% type II muscle fiber) from normal dogs and dogs with tachycardia-induced heart failure. RESULTS: S-nitroso-N-acetylpenicillamine (SNAP), carbachol, and bradykinin at doses of 10(-7) to 10(-4) mol/L concentration significantly suppressed tissue O2 consumption both in the absence and presence of 2,4-dinitrophenol (1 mmol/L), a mitochondrial uncoupler. These effects were not significantly different in the accessory head of the triceps brachii (90% type I muscle fiber) and extensor carpi radialis (86% type II muscle fiber). The effects of carbachol and bradykinin but not SNAP were attenuated by NG-nitro-L-arginine (10(-4) mol/L), indicating inhibition of the formation of endogenous NO. The inhibitory effect on tissue O2 consumption in response to carbachol and bradykinin became significantly smaller in skeletal muscle from dogs with pacing-induced heart failure, but the effects of SNAP were unchanged. CONCLUSIONS: Endogenous NO released from microvascular endothelium may play an important physiologic role in the modulation of cellular respiration in skeletal muscle, and the loss of this regulatory function may contribute to peripheral metabolic disorders and poor exercise tolerance during heart failure.