A DNA vaccine candidate expressing dengue-3 virus prM and E proteins elicits neutralizing antibodies and protects mice against lethal challenge

In an effort to develop a suitable DNA vaccine candidate for dengue, using dengue-3 virus (DENV-3) as a prototype, the genes coding for premembrane (prM) and envelope proteins (E) were inserted into an expression plasmid. After selecting recombinant clones containing prM/E genes, protein expression in the cell monolayer was detected by indirect immunofluorescence and immunoprecipitation assays. After selecting three vaccine candidates (pVAC1DEN3, pVAC2DEN3 and pVAC3DEN3), they were analyzed in vivo to determine their ability to induce a DENV-3-specific immune response. After three immunizations, the spleens of the immunized animals were isolated, and the cells were cultivated to measure cytokine levels by ELISA and used for lymphoproliferation assays. All of the animals inoculated with the recombinant clones induced neutralizing antibodies against DENV-3 and produced a T cell proliferation response after specific stimuli. Immunized and control mice were challenged with a lethal dose of DENV-3 and observed in order to assess their survival capability. The groups that presented the best survival rate after the challenge were the animals vaccinated with the pVAC3DEN3 clones, with an 80% survival rate. Thus, these data show that we have manufactured a vaccine candidate for DENV-3 that is able to induce a specific immune response and protects mice against a lethal challenge.

A DNA vaccine candidate encoding the structural prM/E proteins elicits a strong immune response and protects mice against dengue-4 virus infection

A DNA vaccine expressing dengue-4 virus premembrane (prM) and envelope (E) genes was produced by inserting these genes into a mammalian expression plasmid (pCI). Following a thorough screening, including confirmation of protein expression in vitro, a recombinant clone expressing these genes was selected and used to immunize BALB/c mice. After 3 immunizations all the animals produced detectable levels of neutralizing antibodies against dengue-4 virus. The cytokines levels and T cell proliferation, detected ex vivo from the spleen of the immunized mice, showed that our construction induced substantial immune stimulation after three doses. Even though the antibody levels, induced by our DNA vaccine, were lower than those obtained in mice immunized with dengue-4 virus the levels of protection were high with this vaccine. This observation is further supported by the fact that 80% of the vaccine immunized group was protected against lethal challenge. In conclusion, we developed a DNA vaccine employing the genes of the prM and E proteins from dengue-4 virus that protects mice against this virus. (C) 2010 Elsevier Ltd. All rights reserved.

Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 mu M) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 mu M can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

Bcl-X-L translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

Background. The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X-L, pro-apoptotic Bax and Bad), Methods. Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) Or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (West ern immunoblots, densitometry, immunoelectron microscopy). Results. Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X-L and Bax, but not Bcl-2 or Bad, was identified in control distal cells, Bcl-X-L and Bax had nonsignificant increases (P > 0.05) in these cells. Bcl-2, Bax, and Bcl-X-L, but not Bad, were endogenously expressed in control proximal cells. Bcl-X-L was significantly decreased in treated proximal cultures (P < 0.05), with Bas and Bcl-2 having nonsignificant increases (P > 0.05). Immunoelectron microscopy localization indicated that control and treated hut surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X-L from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-XL expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. Conclusion. The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X-L in proximal cells, as well as translocation of Bcl-X-L protein to mitochondria within the surviving distal cells.

Metalloproteinase inhibition protects against cardiomyocyte injury during experimental acute pulmonary thromboembolism

Objectives: Up-regulated matrix metalloproteinases may be involved in the development of cardiomyocyte injury and the degradation of troponin associated with acute pulmonary thromboembolism. We examined whether pretreatment with doxycycline (a nonspecific matrix metalloproteinase inhibitor) protects against cardiomyocyte injury associated with acute pulmonary thromboembolism. Design: Controlled animal study. Setting: University research laboratory. Subjects: Mongrel dogs. Interventions: Anesthetized animals received doxycycline (10 mg/kg intravenously) or saline and acute pulmonary thromboembolism was induced with autologous blood clots injected into the right atrium. Control animals received doxycycline (or saline). Measurements and Main Results: Hemodynamic measurements were performed, and acute pulmonary thromboembolism increased baseline mean pulmonary arterial pressure and pulmonary vascular resistance by approximately 160% and 362%, respectively (both p<.05), 120 mins after acute pulmonary thromboembolism. Pretreatment with doxycycline attenuated these increases (to 125% and 232%, respectively; both p<.05). Although acute pulmonary thromboembolism tended to increase the right ventricle maximum rate of isovolumic pressure development and the maximum rate of isovolumic pressure decay, doxycycline produced no effects on these parameters. Gelatin zymograms of right ventricle showed that acute pulmonary thromboembolism marginally increased matrix metalloproteinase-9 (but not matrix metalloproteinase-2) levels in the right ventricle. A fluorometric assay to assess net matrix metalloproteinase activities showed that acute pulmonary thromboembolism increased matrix metalloproteinase activities in the right ventricle by >100% (p<.05), and this finding was confirmed by in situ zymography of the right ventricle. Doxycycline attenuated acute pulmonary thromboembolism-induced increases in right ventricle matrix metalloproteinase activities. Acute pulmonary thromboembolism induced neutrophil accumulation in the right ventricle, as estimated by myeloperoxidase activity, and doxycycline blunted this effect (p<.05). Serum cardiac troponin I concentrations, which reflect cardiomyocyte injury, increased after acute pulmonary thromboembolism, and this increase was attenuated by pretreatment with doxycycline (p<.05). Conclusions: We found evidence supporting the idea that acute pulmonary thromboembolism is associated with increased matrix metalloproteinase activities in the right ventricle, which may lead to degradation of sarcomeric proteins, including cardiac troponin I. Inhibition of matrix metalloproteinases may be an effective therapeutic intervention in the management of acute pulmonary thromboembolism. (Crit Care Med 2011; 39: 349-356)

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress. we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201 +/- 5 vs. 163 +/- 7 mm Hg in diabetic rats untreated and treated with lercaniclipine, respectively; P < 0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P < 0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P < 0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P < 0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2MMP-2 expression. (C) 2008 Published by Elsevier B.V.

DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo; however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 mug of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN,viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.

Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as pepstatin A (a CatD inhibitor) increased acetate-induced apoptosis. These results mimicked our previous data in the yeast system showing that acetic acid activates a mitochondria-dependent apoptosis process associated with vacuolar membrane permeabilization and release of the vacuolar protease Pep4p, ortholog of mammalian CatD. Indeed, this protease was required for cell survival in a manner dependent on its catalytic activity and for efficient mitochondrial degradation independently of autophagy. In this study, we therefore assessed the role of CatD in acetate-induced mitochondrial alterations. We found that, similar to acetic acid in yeast, acetate-induced apoptosis is not associated with autophagy induction in CRC cells. Moreover, inhibition of CatD with small interfering RNA or pepstatin A enhanced apoptosis associated with higher mitochondrial dysfunction and increased mitochondrial mass. This effect seems to be specific, as inhibition of CatB and CatL with E-64d had no effect, nor were these proteases significantly released to the cytosol during acetate-induced apoptosis. Using yeast cells, we further show that the role of Pep4p in mitochondrial degradation depends on its protease activity and is complemented by CatD, indicating that this mechanism is conserved. In summary, the clues provided by the yeast model unveiled a novel CatD function in the degradation of damaged mitochondria when autophagy is impaired, which protects CRC cells from acetate-induced apoptosis. CatD inhibitors could therefore enhance acetate-mediated cancer cell death, presenting a novel strategy for prevention or therapy of CRC.

Co-administration of Apelin and T4 Protects Inotropic and Chronotropic Changes Occurring in Hypothyroid Rats

AbstractBackground:One of the most important thyroid hormone targets is the cardiovascular system. Hemodynamic changes, such as decreased resting heart rate (HR), myocardial contractility, and cardiac output, and increased diastolic pressure and systemic vascular resistance, have been observed in hypothyroid patients. Moreover, in these patients, ECG changes include sinus bradycardia and low voltage complexes (P waves or QRS complexes).Objective:This study aimed at evaluating the prophylactic effect of apelin on HR changes and QRS voltage that occur in propylthiouracil (PTU)-induced hypothyroid rats.Method:In this study, 48 adult male Wistar rats weighing 170-235g were randomly divided into 6 groups: Control group (normal saline ip injection + tap water gavage); P group (PTU 0.05%, in drinking water); A group (apelin 200 µg.kg-1.day-1, ip); PA group [co-administration of PTU and apelin]; PT group [co-administration of PTU + T4 (0.2 mg/g per day, gavage)]; and PAT group (co-administration of PTU, apelin and T4). All experiments were performed for 28 consecutive days, and then the animals were anesthetized with an ip injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). Lead II electrocardiogram was recorded to calculate HR and QRS voltage.Results:Heart rate and QRS voltage increased more significantly in the hypothyroid group that consumed both apelin and T4 (201 ± 4 beat/min, 0.71 ± 0.02 mv vs. hypothyroid 145 ± 9 beat/min, 0.563 ± 0.015 mv; respectively).Conclusion:The co-administration of apelin and T4 showed a protective effect on QRS voltage and HR in PTU‑induced hypothyroid rats.

Sumoylated PPARalpha mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice.

As most metabolic studies are conducted in male animals, understanding the sex specificity of the underlying molecular pathways has been broadly neglected; for example, whether PPARs elicit sex-dependent responses has not been determined. Here we show that in mice, PPARalpha has broad female-dependent repressive actions on hepatic genes involved in steroid metabolism and immunity. In male mice, this effect was reproduced by the administration of a synthetic PPARalpha ligand. Using the steroid oxysterol 7alpha-hydroxylase cytochrome P4507b1 (Cyp7b1) gene as a model, we elucidated the molecular mechanism of this sex-specific PPARalpha-dependent repression. Initial sumoylation of the ligand-binding domain of PPARalpha triggered the interaction of PPARalpha with GA-binding protein alpha (GABPalpha) bound to the target Cyp7b1 promoter. Histone deacetylase and DNA and histone methylases were then recruited, and the adjacent Sp1-binding site and histones were methylated. These events resulted in loss of Sp1-stimulated expression and thus downregulation of Cyp7b1. Physiologically, this repression conferred on female mice protection against estrogen-induced intrahepatic cholestasis, the most common hepatic disease during pregnancy, suggesting a therapeutic target for prevention of this disease.

The sirtuin inhibitor cambinol impairs MAPK signaling, inhibits inflammatory and innate immune responses and protects from septic shock.

Sirtuins (SIRT1-7) are NAD(+)-dependent histone deacetylases (HDACs) that play an important role in the control of metabolism and proliferation and the development of age-associated diseases like oncologic, cardiovascular and neurodegenerative diseases. Cambinol was originally described as a compound inhibiting the activity of SIRT1 and SIRT2, with efficient anti-tumor activity in vivo. Here, we studied the effects of cambinol on microbial sensing by mouse and human immune cells and on host innate immune responses in vivo. Cambinol inhibited the expression of cytokines (TNF, IL-1β, IL-6, IL-12p40, and IFN-γ), NO and CD40 by macrophages, dendritic cells, splenocytes and whole blood stimulated with a broad range of microbial and inflammasome stimuli. Sirtinol, an inhibitor of SIRT1 and SIRT2 structurally related to cambinol, also decreased macrophage response to TLR stimulation. On the contrary, selective inhibitors of SIRT1 (EX-527 and CHIC-35) and SIRT2 (AGK2 and AK-7) used alone or in combination had no inhibitory effect, suggesting that cambinol and sirtinol act by targeting more than just SIRT1 and SIRT2. Cambinol and sirtinol at anti-inflammatory concentrations also did not inhibit SIRT6 activity in in vitro assay. At the molecular level, cambinol impaired stimulus-induced phosphorylation of MAPKs and upstream MEKs. Going well along with its powerful anti-inflammatory activity, cambinol reduced TNF blood levels and bacteremia and improved survival in preclinical models of endotoxic shock and septic shock. Altogether, our data suggest that pharmacological inhibitors of sirtuins structurally related to cambinol may be of clinical interest to treat inflammatory diseases.

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65% smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75% in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57% by SC and 49% by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization.

Smad3 deficiency in mice protects against insulin resistance and obesity induced by a high-fat diet.

OBJECTIVE-Obesity and associated pathologies are major global health problems. Transforming growth factor-beta/Smad3 signaling has been implicated in various metabolic processes, including adipogenesis, insulin expression, and pancreatic beta-cell function. However, the systemic effects of Smad3 deficiency on adiposity and insulin resistance in vivo remain elusive. This study investigated the effects of Smad3 deficiency on whole-body glucose and lipid homeostasis and its contribution to the development of obesity and type 2 diabetes.RESEARCH DESIGN AND METHODS-We compared various metabolic profiles of Smad3-knockout and wild-type mice. We also determined the mechanism by which Smad3 deficiency affects the expression of genes involved in adipogenesis and metabolism. Mice were then challenged with a high-fat diet to study the impact of Smad3 deficiency on the development of obesity and insulin resistance.RESULTS-Smad3-knockout mice exhibited diminished adiposity with improved glucose tolerance and insulin sensitivity. Chromatin immunoprecipitation assay revealed that Smad3 deficiency increased CCAAT/enhancer-binding protein beta-C/EBP homologous protein 10 interaction and exerted a differential regulation on proliferator-activated receptor beta/delta and proliferator-activated receptor gamma expression in adipocytes. Focused gene expression profiling revealed an altered expression of genes involved in adipogenesis, lipid accumulation, and fatty acid beta-oxidation, indicative of altered adipose physiology. Despite reduced physical activity with no modification in food intake, these mutant mice were resistant to obesity and insulin resistance induced by a high-fat diet.CONCLUSIONS-Smad3 is a multifaceted regulator in adipose physiology and the pathogenesis of obesity and type 2 diabetes, suggesting that Smad3 may be a potential target for the treatment of obesity and its associated disorders.

Schistosomiasis protects against multiple sclerosis

The incidences of schistosomiasis and multiple sclerosis (MS) are mutually exclusive worldwide suggesting that schistosomiasis may offer protection against the induction of the immune-mediated disease, MS. Recent studies using the mouse model of MS, experimental autoimmune encephalomyelitis, support a direct suppression of the onset of MS by chronic Schistosoma mansoni infection. Self-reactive Th1 but not Th2 responses develop in infected mice immunized with myelin oligodendrocyte glycoprotein albeit at reduced levels indicating that the induction of auto-reactive T cells is not abolished nor phenotypically altered. CNS infiltration by inflammatory cells, particularly macrophages, is significantly reduced in S. mansoni-infected, immunized mice compared to uninfected, immunized mice. Because activated macrophages are crucial to the induction of clinical disease, these findings support the hypothesis that differences in macrophage activation may contribute to the reduced incidence and delayed progression of experimental autoimmune encephalomyelitis during schistosomiasis.

Coating carbon nanotubes with a polystyrene-based polymer protects against pulmonary toxicity.

BACKGROUND: carbon nanotubes (CNT) can have adverse effects on health. Therefore, minimizing the risk associated with CNT exposure is of crucial importance. The aim of this work was to evaluate if coating multi-walled CNT (MWCNT) with polymers could modify their toxicity, thus representing a useful strategy to decrease adverse health effects of CNT. We used industrially-produced MWCNT uncoated (NT1) or coated (50/50 wt%) with acid-based (NT2) or polystyrene-based (NT3) polymer, and exposed murine macrophages (RAW 264.7 cell line) or Balb/c mice by intratracheal administration. Biological experiments were performed both in vitro and in vivo, examining time- and dose-dependent effects of CNT, in terms of cytotoxicity, expression of genes and proteins related to oxidative stress, inflammation and tissue remodeling, cell and lung tissue morphology (optical and transmission electron microscopy), and bronchoalveolar lavage fluid content analysis.RESULTS: extensive physico-chemical characterization of MWCNT was performed, and showed, although similar dimensions for the 3 MWCNT, a much smaller specific surface area for NT2 and NT3 as compared to NT1 (54.1, 34 and 227.54 m(2)/g respectively), along with different surface characteristics. MWCNT-induced cytotoxicity, oxidative stress, and inflammation were increased by acid-based and decreased by polystyrene-based polymer coating both in vitro in murine macrophages and in vivo in lung of mice monitored for 6 months.CONCLUSIONS: these results demonstrate that coating CNT with polymers, without affecting their intrinsic structure, may constitute a useful strategy for decreasing CNT toxicity, and may hold promise for improving occupational safety and that of general the user.