Leishmania major MAPK4 intercepts and redirects CD40 signaling promoting infection.

The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets.

Development and Characterization of an Avirulent Leishmania major Strain.

Leishmania major causes cutaneous leishmaniasis. An antileishmanial vaccine for humans is unavailable. In this study, we report development of two attenuated L. major strains-5ASKH-HP and LV39-HP-by continuous culture (high passage) of the corresponding virulent strains (low passage). Both avirulent strains showed similar changes in proteome profiles when analyzed by surface-enhanced laser desorption ionization mass spectrometry. Liquid chromatography-mass spectrometry and microarray characterization of 5ASKH strains revealed substantially altered gene and protein expression profiles, respectively. Both virulent and avirulent L. major strains grew comparably in culture, but the avirulent strain survived significantly less in BALB/c-derived peritoneal macrophages. Both attenuated strains failed to infect BALB/c mice and elicited IFN-γ, but not IL-4 and IL-10, responses. 5ASKH-HP parasites failed to induce significant infection even in severely immunocompromised- SCID or inducible NO synthase-, CD40-, or IL-12-deficient mice, indicating attenuation. The avirulent strain induced less IL-10, but higher IL-12, in macrophages. The avirulent strain failed to reduce CD40 relocation to the detergent-resistant membrane domain and to inhibit CD40-induced phosphorylation of the kinases Lyn and protein kinase C-ß and MAPKs MKK-3/6 and p38MAPK or to upregulate MEK-1/2 and ERK-1/2 in BALB/c-derived peritoneal macrophages. The virulent and the avirulent strains reciprocally modulated CD40-induced Ras-mediated signaling through PI-3K and Raf-1. Avirulent 5ASKH-primed BALB/c mice were protected against virulent L. major challenge infection. The loss of virulence accompanied by substantially altered proteome profiles and the elicitation of host-protective immune responses indicate plausibly irreversible attenuation of the L. major strain and its potential use as a vaccine strain.

Dietary fat proportionately enhances oxidative stress and glucose intolerance followed by impaired expression of the genes associated with mitochondrial biogenesis.

Consumption of food that surpasses the metabolic necessity of the body leads to an epidemic condition termed obesity, which causes several metabolic disorders including oxidative damage. Dietary intervention can enlighten the mechanisms and therapeutics associated with these metabolic disorders. The reported studies related to diet include fat of different kinds and from different sources, however they lack dose response aspects. Our study highlighted the importance of dietary fat modification in modulating oxidative stress-induced glucose intolerance. Animals were maintained on a diet with a varied content of fat (30%/45%/60%) for 12 weeks and the 'withdrawal' group was fed a standard diet for another 10 weeks. The diet containing 60 energy% of fat displayed glucose intolerance, high ALT, low GSH levels and tissue-specific modulation of the prooxidant/antioxidant enzymatic activities in the liver/muscles. Prolonged sustenance of the 60 energy% fat containing diet-fed rats on standard diet led to the alteration of antioxidant activities, reversing the oxidative damage. Notably, the 'withdrawal' group displayed an organ-specific response towards dietary modification where the recovery of the antioxidant activities was observed to be much more pronounced in the liver as compared to the muscle. Further, we identified the differential expression of liver/muscle-specific genes associated with oxidative stress and mitochondrial biogenesis in response to the differing fat content. These genes can serve as markers for HFD-induced metabolic complications involving the liver/muscle. Altogether, our study has highlighted the novel area where obesity-induced oxidative stress linked alterations expressed diet and organ specific responses that are recovered by altering the dietary regimen. Future investigation of dietary modulation will open nascent avenues for developing therapeutic modalities addressing obesity-related metabolic complications.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9.

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 µg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

Mechanisms of isoproterenol-induced cardiac mitochondrial damage: protective actions of melatonin.

Mitochondrial dysfunction due to oxidative damage is the key feature of several diseases. We have earlier reported mitochondrial damage resulting from the generation of oxidative stress as a major pathophysiological effect of isoproterenol (ISO)-induced myocardial ischemia in rats. That melatonin is an antioxidant that ameliorates oxidative stress in experimental animals as well as in humans is well established. We previously demonstrated that melatonin provides cardioprotection against ISO-induced myocardial injury as a result of its antioxidant properties. The mechanism of ISO-induced cardiac mitochondrial damage and protection by melatonin, however, remains to be elucidated in vitro. In this study, we provide evidence that ISO causes dysfunction of isolated goat heart mitochondria. Incubation of cardiac mitochondria with increasing concentrations of ISO decreased mitochondrial succinate dehydrogenase (SDH) activity, which plays a pivotal role in mitochondrial bioenergetics, as well as altered the activities of other key enzymes of the Kreb's cycle and the respiratory chain. Co-incubation of ISO-challenged mitochondria with melatonin prevented the alterations in enzyme activity. That these changes in mitochondrial energy metabolism were due to the perpetration of oxidative stress by ISO was evident from the increased levels of lipid peroxidation and decreased reduced glutathione/oxidized glutathione ratio. ISO-induced oxidative stress also altered mitochondrial redox potential and brought about changes in the activity of the antioxidant enzymes manganese superoxide dismutase and glutathione peroxidase, eventually leading to alterations in total ATPase activity and membrane potential. Melatonin ameliorated these changes likely through its antioxidant abilities suggesting a possible mechanism of cardioprotection by this indole against ISO-induced myocardial injury.

Melatonin protects against isoproterenol-induced alterations in cardiac mitochondrial energy-metabolizing enzymes, apoptotic proteins, and assists in complete recovery from myocardial injury in rats.

The present study was undertaken to explore the protective effect of melatonin against isoproterenol bitartrate (ISO)-induced rat myocardial injury and to test whether melatonin has a role in preventing myocardial injury and recovery when the ISO-induced stress is withdrawn. Treatment for rats with ISO altered the activities of some of the key mitochondrial enzymes related to energy metabolism, the levels of some stress proteins, and the proteins related to apoptosis. These changes were found to be ameliorated when the animals were pretreated with melatonin at a dose of 10 mg/kg BW, i.p. In addition to its ability to reduce ISO-induced mitochondrial dysfunction, we also studied the role of melatonin in the recovery of the cardiac tissue after ISO-induced damage. Continuation of melatonin treatment in rats after the withdrawal of ISO treatment was found to reduce the activities of cardiac injury biomarkers including serum glutamate oxaloacetate transaminase (SGOT), lactate dehydrogenase (LDH), and cardio-specific LDH1 to control levels. The levels of tissue lipid peroxidation and reduced glutathione were also brought back to that seen in control animals by continued melatonin treatment. Continuation of melatonin treatment in post-ISO treatment period was also found to improve cardiac tissue morphology and heart function. Thus, the findings indicate melatonin's ability to provide cardio protection at a low pharmacological dose and its role in the recovery process. Melatonin, a molecule with very low or no toxicity may be considered as a therapeutic for the treatment for ischemic heart disease.

Protective effect of aqueous Curry leaf (Murraya koenigii) extract against cadmium-induced oxidative stress in rat heart.

Treatment of rats with a low dose of cadmium chloride caused a significant damage in the rat cardiac tissue indicated by the increase in the level of serum glutamate oxaloacetate transaminase and lactate dehydrogenase1 activities. Histological studies confirmed the damage due to cadmium. That cadmium-induced tissue damage was caused due to oxidative stress was evident from the changes observed in the levels of lipid peroxidation and reduced glutathione, the protein carbonyl content, and the alterations in the activities of cardiac antioxidant and pro-oxidant enzymes. Treatment of rats with cadmium also caused alterations in the activities of mitochondrial Kreb's cycle as well as respiratory chain enzymes. All these changes were ameliorated when the rats were pre-treated with an aqueous extract of Curry leaf (Murraya koenigii). The studies indicated that the aqueous extract of Curry leaf protects the rat cardiac tissue against cadmium-induced oxidative stress possibly through its antioxidant activity. As curry leaf is consumed by people as part of their diet in India and South-East Asian and some European countries as well, and, as it has no reported side-effects, the results seem to have relevance at places where humans are exposed to cadmium environmentally or occupationally.

Protective effect of aqueous garlic extract against lead-induced hepatic injury in rats.

Effect of aqueous extract of garlic on hepatic injury due to lead-induced oxidative stress in experimental rats has been investigated. Lead acetate (LA) at a dose of 15 mg/kg body wt was administered ip to rats for 7 consecutive days to induce hepatic injury. Freshly prepared aqueous garlic extract (AGE) at a dose of 50 mg/kg body wt was fed orally to rats 1 h before LA treatment for similar period. LA treatment caused hepatic injury as evident from increased activities of serum glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP), increased serum bilirubin level and damage in the tissue morphology. Lead-induced oxidative stress in liver was evident from increased levels of lipid peroxidation and reduced glutathione. The decreased activity of superoxide dismutase (SOD) and an increased activity of catalase as well as an increased activity of xanthine oxidase (XO) indicate generation and possible accumulation of reactive oxygen intermediates. Furthermore, altered activities of lactate dehydrogenase (LDH), isocitrate dehydrogenase (ICDH), alpha-keto glutarate dehydrogenase (alpha-KGDH) and succinate dehydrogenase (SDH) also indicate an impaired substrate utilization and generation of oxidative stress. All these changes were found to be mitigated when the rats were pre-treated with the AGE. Results indicate that AGE has the potential to ameliorate lead-induced hepatic injury due to oxidative stress in rats. The protective effects may be due to the antioxidant properties of AGE and may have future therapeutic relevance.

Melatonin protects against isoproterenol-induced myocardial injury in the rat: antioxidative mechanisms.

The present study was undertaken to explore the protective effect of melatonin against isoproterenol bitartrate (ISO)-induced myocardial injury in rat. Treatment of rats with ISO increased the level of lipid peroxidation products and decreased the reduced glutathione levels in cardiac tissue indicating that this synthetic catecholamine induces oxidative damage following oxidative stress. Pretreatment of ISO-injected rats with melatonin at a dose of 10 mg/kg body weight, i.p. prevented these changes. Additionally, melatonin also restored the activities and the levels of antioxidant enzymes which were found to be altered by ISO treatment. Treatment of rats with ISO resulted into an increased generation of hydroxyl radicals with melatonin pretreatment significantly reducing their production. Finally, treatment of rats with ISO caused a lowering of systolic pressure with reduced cardiac output and diastolic dysfunction whereas melatonin pretreatment significantly restored many of these parameters to normal. The findings document melatonin's ability to provide cardio protection at a low pharmacological dose. Melatonin has virtually no toxicity which raises the possibility of this indole being a therapeutic treatment for ischemic heart disease.

Excess of glucocorticoid induces cardiac dysfunction via activating angiotensin II pathway.

BACKGROUND: Glucocorticoid is widely used as an anti-inflammatory drug in various diseases however excess of it often causes cardiovascular complications. The present study was undertaken to understand the molecular mechanism of glucocorticoid-induced cardiac dysfunction. METHODS: Rats were treated daily with synthetic glucocorticoid, dexamethasone with or without mifepristone or losartan up to 15 days. Hemodynamic parameters were measured by PV-loop method using Millar's instrument. Cardiac remodelling, fibrosis and oxidative stress were monitored after 15 days. RESULTS: The systolic blood pressure was increased whereas the heart beat and cardiac output (n=6) were decreased by dexamethasone. Dexamethasone caused increase in the heart weight to body weight ratio (P<0.001, n=20), increased level of mRNA of atrial natriuretic peptide and an increased deposition of collagens in the extracellular matrix of the left ventricle which were inhibited by both mifepristone and losartan. The rate of oxygen consumption was decreased in association with increased levels of hypoxia inducible factor 1alpha, lipid peroxidation (P<0.01, n=3) and superoxide dismutase activity (P<0.01, n=3) in dexamethasone treated rat heart. All these changes were reversed by mifepristone and losartan. CONCLUSIONS: The excess of glucocorticoid induces cardiac remodelling and pathophysiolgical changes of the myocardium via angiotensin II signalling pathway.