Day 2 :
You Yang Zhao
Northwestern Feinberg School of Medicine, USA
Keynote: Enhancing endothelial regeneration and vascular repair for treatment of sepsis and acute respiratory distress syndrome
Time : 09:30-10:10
You Yang Zhao is the William G Swartchild’s Jr. Distinguished Research Professor and Program Director for Lung and Vascular Biology at the Ann & Robert H Lurie Children’s Hospital of Chicago, and Department of Pediatrics at Northwestern University Feinberg School of Medicine. He received his training in cardiopulmonary vascular biology at Harvard University and UCSD. Prior to his tenure at LCH, he was a Professor at the Department of Pharmacology at the University of Illinois at Chicago and Senior Research Scientist in Cardiovascular Drug Discovery in Pharmacia/Pfizer Inc. His research is focused on lung and vascular biology to delineate the molecular mechanisms of endothelial regeneration and resolution of inflammatory injury, as well as pulmonary vascular remodeling in the pathogenesis of pulmonary arterial hypertension (PAH), and thereby to provide novel druggable targets and therapeutic strategies for treatment of acute respiratory distress syndrome and PAH. He has published many papers in top-tier journals such as Nat Med, PNAS, J Clin Invest, J Exp Med and Circulation. His lab is well-funded with multiple R01 grants and PPG grant from NIH.
Statement of the Problem: Evidence from human and animal studies has shown the key role of microvascular leakage in determining the outcome of sepsis and acute respiratory distress syndrome (ARDS). However, little is known about the signaling pathways regulating endothelial regeneration and vascular repair following sepsis challenge, and hence no crucial druggable targets identified yet for development of effective drug(s) and the mortality rate remains as high as 40%.
Methodology & Theoretical Orientation: Employing genetic lineage tracing mice to define the cell origin of endothelial regeneration responsible for vascular repair. Various genetically modified mouse models as well as pharmacological approach were used to identify the transcriptional factors and underlying signaling pathways mediating endothelial regeneration.
Findings: Employing a genetic lineage tracing approach, here we show that resident endothelial cell is the origin of endothelial regeneration in mouse lungs after lipopolysaccharide-induced inflammatory injury. Mice with Tie2Cre-mediated disruption of FoxM1 in endothelial cells exhibited impaired endothelial regeneration and vascular repair and thus the forkhead transcriptional factor FoxM1 is the critical TF for endothelial regeneration. Employing pharmacological inhibitors, we demonstrate that endothelial regeneration selectively requires activation of p110γPI3K signaling, which thereby mediates the expression of the endothelial reparative transcription factor FoxM1. We further identified SDF-1a as the critical agonist to activate the GPCR-dependent p110gPI3K in EC through CXCR4 and thereby induced FoxM1-dependent endothelial regeneration. We also observed diminished expression of p110g in pulmonary vascular ECs of ARDS patients associated with severe sepsis, suggesting that impaired p110g-FoxM1 endothelial regeneration and vascular repair signaling pathway is a critical factor in persistent leaky lung microvessels and edema formation in the disease. In aged mice, we observed defective endothelial regeneration and vascular repair which was caused by impaired p110g-FoxM1 signaling. We will discuss the pharmacological approach to activate this intrinsic regenerative pathway in aged lungs to restore vascular integrity and promote survival following sepsis challenge.
Conclusion & Significance: We identify endothelial p110g-FoxM1 signaling axis as the critical mediator of endothelial regeneration and vascular repair following sepsis challenge. Activation of this intrinsic regenerative pathway may represent a novel strategy for the treatment of severe sepsis and ARDS.
A J Mixson
University of Maryland School of Medicine, USA
Keynote: Targeted RNAi therapeutics with an optimized HK carrier
A J Mixson has been working in the Pathology Department at the University of Maryland, School of Medicine since 1994 with an initial focus on non-viral delivery of anti-angiogenic nucleic acids (plasmids, siRNA, DNAzymes) with liposomes utilizing either in vitro or in vivo model systems. He has been awarded several US and European patents on gene therapy and anti-angiogenesis.
Statement of the Problem: The development of non-viral carriers for clinical studies has been painstakingly slow. Histidine-rich peptide mirror many of the properties of polyethylenimine (PEI) as carriers of nucleic acids, in that they contain both cationic and endosomal releasing components to increase transfection. Although these peptide carriers are less toxic than PEI, toxicity including cytokine induction and transfection efficiency in vivo may limit the utility of these peptide carriers.
Methods: Solid phase methods was used for the synthesis of HK peptides that varied in their sequence patterns and branching. Silencing assays assessed the ability of the HK carrier to decrease luciferase or the Raf-1 oncogene in tumors cells or in tumor xenografts. Modifications of the peptide by pegylation and ligand (RP) were compared to unmodified HK peptides in pharmacokinetic and silencing studies. Multiplex bead-based and ELISA assays measured cytokine levels.
Findings: With non-invasive imaging, systemically administered surface-modified RP HK:siRNA nanoplexes showed nearly 4-fold greater blood levels, 40% higher accumulation in tumor tissue and 60% lower luciferase activity than unmodified HK:siRNA nanoplexes (Figure 1). We then determined whether the surface-modified RP HK:siRNA nanoplex was more effective in reducing MDA-MB-435 tumor growth with an siRNA targeting Raf-1. Repeated systemic administration of the selected surface-modified RP HK:siRNA nanoplexes targeting Raf-1 showed 45% greater inhibition of tumor growth than unmodified HK:siRNA nanoplexes and 90% greater inhibition of tumor growth than untreated mice. Cytokine induction was minimal with the modified HK polyplexes compared to the unmodified polyplexes.
Conclusions: The results show that targeted HK:siRNA nanoplexes gave a significantly greater reduction of the targeted gene expression in the tumor. This resulted in marked decrease in tumor growth rate with histochemistry measurements of downstream effects supporting siRNA-mediated mechanism of action for the observed efficacy.
- Plenary Talk
Yalavarthy Prameela Devi
Kakatiya University, India
Title: Toxic chemicals in personal care products and their hazards with special reference to Triclosan
Yalavarthy Prameela Devi has obtained her MSc in Zoology with Environmental Biology specialization and PhD in Environmental Biology from S V University, India in 1984. She is presently working as the Dean, Faculty of Sciences at Kakatiya University, India. She has 33 years of teaching and 37 years of research experience and has good knowledge in Environmental Biotechnology and Environmental Bioanalytical Techniques. She is an Endeavour Fellow of Australia 2010. She has published nearly 110 scientific research papers and presented over 100 talks at various national and international seminars, workshops, training programs and conferences. She has received UGC Research Award, India in 2006. Her research interests include the development of low cost field kits for environmental monitoring of toxic chemicals using biological principles, environmental toxicology, ecological restoration and environmental management and global climate change. She is an Honorary Adviser to many environmental consultancy service companies in India.
Personal care products refer to a wide variety of items that are found in the health and beauty sections of drug and departmental stores. Some of these products are cosmetics which includes skin moisturizers, perfumes, lipsticks, fingernail polishes, eye and facial makeup preparations, shampoos, hair colors, toothpastes and deodorants and also covers some drugs like skin protectants (lip balms and diaper ointments), mouthwashes marketed with therapeutic claims, antiperspirants and treatments for dandruff or acne. Most personal care products on the market contain toxic chemicals like triclosan, parabens, phthalates, synthetic colors, fragrance, sodium lauryl sulfate (SLS), formaldehyde and toluene. These chemicals linked to hormonal imbalance, reproductive health problems and even cancer. Many of these chemicals in personal care products have never been tested for safety and may also accumulate and interact in potentially harmful ways. The load adds up quickly day after day as we swallow, breathe in and lather up and these toxins enter into our bodies and may have lasting impact. Many people may not realize that ingredients in cosmetics and personal care products can enter into the environment and harm the environment and may have adverse effects on plants animals and humans. Sorption and photolytic degradation mitigate the availability of these chemicals to aquatic biota and the byproducts of such chemicals may be more resistant to degradation and have higher toxicity than the parent compound. We have studied the toxic effects of Triclosan, a high production volume ingredient used as a bactericide in personal care products such as toothpaste and deodorant, on bacteria, zooplankton and an aquatic fish, Channa punctatus, which is a very common edible fish in south India. We have selected these organisms as these are important in the ecosystem function and any possible damage to these organisms will have an adverse effect in maintaining the balance of the ecosystem. Acute toxicity evaluation of fish on LC50, behavioral changes, biochemical and molecular alterations, histopathological changes were studied. Effects of triclosan on bacterial activity and on zooplankton were also evaluated. The result indicate that triclosan exerts a marked influence on the parameters tested which indicate that if triclosan is discharged into the environment at high levels, the possible destruction of the balance of the ecosystem is expected.
- Molecular Toxicology | Clinical Toxicology | Genetic Toxicology
A J Mixson
University of Maryland School of Medicine, USA
Universidad de Buenos Aires, Argentina
Title: Organochlorine pesticides and breast cancer: Mechanism of action on human breast cancer cells and animal models
Andrea Randi has her expertise in evaluation of pesticides mechanisms of action that act as endocrine disruptors, specifically those ligand compounds of the aryl hydrocarbon receptors (AhR). In her laboratory, she is developing two main lines of work, one related to the induced effects by these compounds in the mammary gland and another in the uterus. In mammary gland, her interest is to investigate the different processes of breast cancer progression, such as proliferation, angiogenesis, migration, invasion and metastasis, analyzing the signaling pathways related with growth factor receptors, AhR and estrogen receptors. On the other hand, in the uterus, she is engaged in analyzing the effects on the development of endometriosis, evaluating the growth of endometriotic lesions, proliferation, migration, invasion and angiogenesis.
Given the number of women affected by breast cancer, considerable interest has been raised in understanding the relationships between environmental chemicals and disease development. Hexachlorobenzene (HCB) is a widespread organochlorine pesticide detected in mother’s milk and lipid foods. HCB is a dioxin-like compound that weakly binds to the aryl hydrocarbon receptor (AhR), a transcription factor that regulates gene expression associated with proliferation, angiogenesis, migration and invasion. We previously demonstrated that HCB acts as an endocrine disruptor in rat mammary gland and an inducer of cell proliferation. Our studies using negative estrogen receptor alpha (-ERα) MDA-MB-231 human breast cancer cells showed that HCB increases cell migration and invasion and enhances tumor growth stimulating lung and liver metastasis in mice breast cancer models. Increasing evidence indicates that transforming growth factor-β1 (TGF-β1) can contribute to tumor maintenance and progression. In a recent investigation, we found that HCB increases TGF-β1 protein levels and activation, as well as Smad3, JNK and p38 phosphorylation in MDA-MB-231. Real time-qPCR results indicated that HCB reduces AhR mRNA expression through TGF-β1 signaling but enhances TGF-β1 mRNA levels involving AhR. HCB enhances cell migration and invasion through the Smad, JNK and p38 pathways. These results demonstrate that HCB modulates the crosstalk between AhR and TGF-β1 and consequently exacerbates a pro-migratory phenotype in this cell line. Finally, HCB induces the angiogenic switch and increases vascular endothelial growth factor (VEGF) expression in a xenograft model with MDA-MB-231. Human microvascular endothelial cells exposed to HCB showed an increase in cyclooxygenase-2 (COX-2), VEGF and AhR expression. HCB induces cell migration and neovasculogenesis in an AhR, COX-2 and VEGF receptor 2-dependent manner. Altogether, the results showed that HCB is able to modulate several breast cancer-related processes in vitro and in vivo and suggest that HCB may be a risk factor for human breast cancer progression.
National Tsing Hua University, Taiwan
Title: Transcriptomic gene-network analysis for neurotoxicity and drug repurposing
Chun-Yu Chuang focuses her research on biological effects of environmental factors on gene expression relevant to diseases.
Our study established a research scheme of integrative transcriptomic analysis for investigating the neurodegenerative toxicity of silver nanoparticles (AgNPs) and applying to explore drug repurposing of spinal muscular atrophy (SMA). The transcriptomic analysis of mouse brain neural cells after AgNPs exposure found that the gene expressions of C-X-C motif chemokine 13 (CXCL13), macrophage receptor with collagenous structure (MARCO) and glutathione synthetase (GSS) were induced for inflammatory response and oxidative stress, and additionally amyloid precursor protein (APP) was induced and neprilysin (NEP) and low-density lipoprotein receptor (LDLR) were reduced for Aβ plaque production and aggregation. It suggested AgNPs could alter gene expressions of Aβ deposition potentially to cause neurodegenerative disorder (Alzheimer's disease) progression underlying Aβ deposition. On the other hand, the 39 human microarray datasets across different types of SMA tissues were used for the integrative transcriptomic analysis, which identified TNFα-BMP4-SERPINE1-GATA6 pathway associated with disease severity. Down-regulation of bone morphogenetic protein 4 (BMP4) may be one of the key points in SMA pathogenesis. BMP4 expression can be induced by cholesterol lowering drug Atorvastatin. SMA mice receiving Atorvastatin treatment prolonged the lifespan, increased body weight, improved motor coordination and exhibited reduced motor neuron degeneration and muscle and cardiac atrophy. In conclusion, the integrative transcriptomic analysis is a useful tool for toxicological effect evaluation and drug repurposing.
Kakatiya University, India
Title: Biochemical and enzymatic changes induced by Butachlor during sublethal exposures in freshwater fish Labeo rohita (Hamilton)
K Prasuna has completed her BSc and MS from Kakatiya University, India. Presently, she is pursuing her PhD at the same university. She has worked as an Associate Pathologist in Clinical Laboratory at Saudi Arabia for about 15 years. Her research interests include environmental toxicology, study of medicinal plants and isolation of bio active compounds for drug development. Currently, she is working on extraction of medicinal plants for isolation and characterization of bioactive compounds to test for anti-venom properties especially for testing the inhibitory activity of toxic proteins.
Statement of the Problem: The need for higher production of food makes us to use pesticides for disease control in both plants and animals. Pesticides usage will harm to the non-target organisms also in the ecosystem. Fish species are very sensitive to various chemicals and will have deleterious effects on the growth and reproduction. The present study was conducted to investigate the effect of acute toxicity of Butachlor, an herbicide, on biochemical alterations and enzymatic inhibition of Indian major carp, Labeo rohita.
Methodology & Theoretical Orientation: The median lethal concentration (LC50) value of the toxicant was estimated by using Finney’s Probit Analysis. The biochemical and enzymatic parameters were estimated spectrophotometrically using standard methods. The LC50 of Butachlor was found to be 0.85 mg/l for 96 hours. Fish were exposed to sublethal doses for 1 day and 10 days to evaluate the biochemical alterations and enzymatic inhibition.
Findings: During sublethal exposures, a significant decrease in glycogen, protein, lipid, carbohydrate and free amino acid (FAA) content was observed. An increase in the level of lactate dehydrogenase (LDH) activity was noticed and a significant decrease in levels of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activity were observed in pesticide exposed tissues of gill, liver, kidney, muscle, intestine and brain when compared with the control tissues. The biochemical alterations might be due to the disruption of internal organs. Increase in enzyme activity may be due to the damage caused to the lysosomal membrane, thus permitting the leakage of lysosomal enzyme into cytosol.
Conclusion & Significance: Decrease in enzyme activity infers to energy metabolism of the nervous system, arrest the transmission of nerve impulses and thereby causing behavioral changes. Thus the above parameters could be effectively used as potential biomarkers of the pesticide toxicity to the fishes.
Kirti Satheesh Pawar
Giriraj Hospital and Intensive Care Unit, India
Title: Revisiting antidotes in the treatment of oraganophosphorus poisoning
Kirti Satheesh Pawar is a Critical Care and Pain Consultant and has expertise in the management of organophosphorus poisoning since 20 years. She has been invited for many national and international clinical meetings to discuss her research and experience in the field of Organophosphorus Poisoning. She is keen to understand controversies in the treatment of organophosphorus poisoning by re-analyzing clinical evidence in this field. She is honored with many awards for her outstanding contribution in the medical field. She believes in ethical medical practice and updating medical knowledge with clinical relevance.
Statement of the Problem: Treatment of Organophosphorus Poisoning (OPP) is a Global challenge. 20% death rate in OPP cases is tragic. Standard treatment for OPP is to give intravenous atropine and pralidoxime. Role of oximes needs more clinical evidence and is need of time. We studied causes of success and failure of oxime therapy in OPP cases and tried to revisit an antidote, atropine, for its extrapolated role in OPP cases since decades.
Methodology & Theoretical Orientation: Atropine is a muscarinic antidote to treat the muscarinic crisis. Oximes reactivate inhibited acetylcholinesterase by organophosphates and checks uninhibited action of acetylcholine at neuromuscular junction. We studied major clinical trials and studies of oximes till date to understand factors affecting success or failure of oxime therapy as well as revisited atropine therapy to understand its effect on outcome.
Findings: WHO recommended minimum dose of pralidoxime as a baseline while treating patients of moderately severe OP poisoning. Unfortunately in last 20 years only two randomized controlled trials (RCT’s) followed this regimen. Use of suboptimal dosage of oximes is a major cause of its ineffectiveness. Inadequacy of respiratory support system could be a major drawback. Failure to maintain optimum oxime concentration as long as active organophosphates are inhibiting acetylcholinesterase is an additional reason for ineffectiveness of oximes. Expenses of pralidoxime is also limiting factor for its liberal use.
Conclusion & Significance: World needs evidence for use of oxime in OPP cases. RCTs to be conducted in future should strictly follow WHO recommended minimum dose of oximes and to be conducted in intensive care unit where cardiorespiratory support system is adequate. An affordable pralidoxime preparation should be a part of public health response. We researchers strongly believe the drug pralidoxime will save many lives and can substantially reduce mortality in OPP cases.
- Young Researchers Forum
Blake R Rushing
Brody School of Medicine at East Carolina University, USA
Title: Detoxification of aflatoxin B1 using dietary molecular species
Blake R Rushing is a PhD Candidate in the Department of Pharmacology and Toxicology at the Brody School of Medicine at East Carolina University, USA.
Aflatoxin B1 (AFB1) is a class 1 carcinogen and a common food contaminant worldwide. It is also a major cause of the development of hepatocellular carcinoma (HCC), making dietary exposure to this toxin very concerning. Existing strategies to reduce AFB1 exposure are limited and as a result, many people are exposed to this toxin worldwide. Issues with current detoxification strategies include harmful byproduct formation, incomplete removal, or the requirement of sophisticated infrastructures. Our study aims to develop a new chemical treatment process to modify AFB1 into a non-carcinogenic form using benign reagents found in human diets. Our strategy targets themutagenic site of the AFB1 molecule, the 8,9-double bond, by adducting it to selected amino acids in dietary proteins.Identification and quantification of aflatoxins was performed using high performance liquid chromatography-electrospray ionization-time of flight mass spectrometry (HPLC-ESI-TOFMS). Optimization of AFB1 hydration was carried out by incubating in various organic acids as well as increasing temperature. Newly formed AFB2a was introduced to alkaline solutions containing amino acids, peptides, and other biological molecules. Products were identified based on changes in retention times and accurate mass values. Mutagenicity of the resulting adduct was determined using an Ames’ test with and without the presence of hepatic microsomes. This study provides a basis for developing a safe and effectivedetoxification method for contaminated foods, reducing exposure to AFB1 worldwide.
Coventry University, UK
Title: Using the human trabeculae muscle work loop assay for assessing cardiac contractility effects of test compound
Mayel Gharanei has completed his PhD on the investigation of the cardiotoxicity of Doxorubicin and strategies for adjunctive cardioprotection. During his PhD, he worked on the optimization of the cardiac work loop technique for safety assessment of pharmaceutical compounds. During his Post-doctoral research, he undertook dedicated studies on the development and validation of the cardiac rat papillary muscle and human trabeculae muscle work loop technique. He is working as the Lead Research Scientist at InoCardia where he supervises research staff and projects associated with testing pharmaceutical compounds on rat papillary and human trabeculae muscle mechanics. As a Research Fellow at Coventry University, he is supervising PhD and Masters by Research students in the field of cardiotoxicity, myocardial ischemia reperfusion injury, cardioprotection, obesity, aging and development and validation of novel clinically relevant cardiovascular non-clinical assays.
Adverse drug response is a significant risk to human health and is costly to the pharmaceutical industry when compounds are withdrawn from market. In vitro cardiac safety testing is generally conducted using predominantly animal tissue or cells or stem cell derived cardiac myocytes. Although side effects of drugs can be caused by many things, one area of great concern is the effects of drugs on the force that heart muscle can produce during its role in pumping blood around the body. Development of a human heart contractility assay would greatly improve the understanding of the human relevance of non-clinical findings; a chemical might cause a change in cardiac contractility in animals but not humans and vice versa. The pharmaceutical, biotechnology, cosmetic, agrochemical, food industries and regulators require an improved assessment of cardiovascular liability associated with drug/chemical-induced changes in cardiac contractility that is more predictive than existing assays. We have demonstrated that the work loop cardiac contractility assay is 93% predictive of human findings than existing assays when using animal tissue. We have expanded this investigation to determine whether the human cardiac work loop assay had the potential to provide a more predictive model of heart muscle dynamics to assess inotropic effects. To validate this assay, a range of positive and negative inotropic agents were tested and effective concentrations compared with those tested in the clinic. The human cardiac muscle work loop assay predicted inotropic effects at clinically relevant concentrations. The human cardiac work loop assay is a new approach to the detection of drug effects on cardiac contractility, providing a superior predictivity of inotropy assessment and importantly identifying inotropy risk at clinically relevant concentrations. The use of human tissue has the potential to replace the use of animals and provide greater concordance with man.
Wright State University Boonshoft School of Medicine, USA
Title: Novel cell-killing mechanisms of hydroxyurea and the implication towards combination therapy for the treatment of fungal infections
Amanpreet Singh is a Post-doctoral Fellow at Mycology Lab, Wadsworth Center, New York State Department of Health, USA. He has obtained his PhD from Department of Biochemistry and Molecular Biology from Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA. His research interests comprise understanding the molecular mechanism of DNA replication checkpoint pathway and fungal pathogenesis.
Mutations in the hem13-1 (heme synthesis) and erg11-1 (ergosterol biosynthesis) pathways genes significantly sensitize the fission yeast Schizosaccharomyces pombe to the ribonucleotide reductase inhibitor hydroxyurea (HU). Our results have also shown that treatment with small molecule inhibitors of the enzyme Erg11 and heme biosynthesis phenocopies the erg11-1 and hem13-1 mutants in sensitizing wild type cells to HU. HU interacts synergistically with several Erg11 inhibitors and the heme biosynthesis inhibitor sampangine in suppressing cell growth or inducing cell death in S. pombe. Importantly, the synergistic drug interactions are likely conserved in eukaryotes because similar synergism has been seen observed in phylogenetically divergent yeast Saccharomyces cerevisiae and the opportunistic fungal pathogen Candida albicans. Based on our genetic data in S. pombe, combinations of sampangine with Erg11 inhibitors are found to be remarkably synergistic in inducing the cell death in C. albicans. Together, these results strongly suggest that HU, sampangine and the Erg11 inhibitors can be further developed in drug combinations for the treatment of fungal infections or other diseases such as cancer. Our results suggests that the combination therapy has the potential to tackle various issues associated with single drug therapy such as toxicity to the hosts, less therapeutic effect, narrower spectrum of activity and more importantly, the development of drug resistance.
Muhammad Ibrar Khan Afridi
Near East University, Turkey
Title: Role of adiponectin in menopausal women
Adipogenesis refers to the differentiation of pre-adipocytes into mature fat cells, i.e., the development of adipose tissue, which varies according to sex and age. Adipocytes differentiate from stellate or fusiform precursor cells of mesenchymal origin. Adiponectin has been postulated to act an important role in the modulation of glucose and lipid metabolism in insulin-sensitive tissue in both humans and animals. The transition from pre to post menopause is associated with the emergency of many features of metabolic state. The intraabdominal body fat increases, low density lipoprotein and triglyceride levels increase while high density lipoprotein decreases. In our study, we aimed to study the changes in adiponectin and anthropometric parameters after menopause. For this purpose the ELISA methods was used in the study to evaluate the values of adiponectin. A total of 70 female in menopause and 90 control subjects were included in this study. The results showed that adiponectin, BMI and blood pressure increased with menopause and in order to investigate the effect of menopause on these parameters, further work must be carried out in the near future.