Aug
12
Mon
2013
Invited Talk: A Far- Western Clinical Proteomics Approach to Detect Molecules of Clinical and Pathological Significance in the Neurodegenerative Disease Multiple Sclerosis @ Amriteshwari Hall
Aug 12 @ 11:27 am – 11:50 am

krishnakumarKrishnakumar Menon, Ph.D.
Associate Professor, Centre for Nanosciences & Molecular Medicine, Amrita University, Kochi, India


A Far-Western Clinical Proteomics Approach to Detect Molecules of Clinical and Pathological Significance in the Neurodegenerative Disease Multiple Sclerosis.

Multiple Sclerosis (MS), an autoimmune neurodegenerative disorder of the central nervous system. The disease affects young adults at their prime age leading to severe debilitation over several years.  Despite advances in MS research, the cause of the disease remains elusive. Thus, our objective is to identify novel molecules of pathological and diagnostic significance important in the understanding, early diagnosis and treatment of MS. Biological fluids such as cerebrospinal fluid (CSF), that bathe the brain serve as a potential source for the identification of pathologically significant autoantibody reactivity in MS.  In this regard, we report the development of an unbiased clinical proteomics approach for the detection of reactive CSF molecules that target brain proteins from patients with MS. Proteins of myelin and myelin-axolemmal complexes were separated by two-dimensional gel electrophoresis, blotted onto membranes and probed separately with biotinylated unprocessed CSF samples. Protein spots that reacted specifically to MS-CSF’s were further analyzed by matrix assisted laser desorption ionization-time-of-flight time-of-flight mass spectrometry. In addition to previously reported proteins found in MS, we have identified several additional molecules involved in mitochondrial and energy metabolism, myelin gene expression and/or cytoskeletal organization. Among these identified molecules, the cellular expression pattern of collapsin response mediator protein-2 and ubiquitin carboxy-terminal hydrolase L1 were investigated in human chronic-active MS lesions by immunohistochemistry. The observation that in multiple sclerosis lesions phosphorylated collapsin response mediator protein-2 was increased, whereas Ubiquitin carboxy-terminal hydrolase L1 was down-regulated, not only highlights the importance of these molecules in the pathology of this disease, but also illustrates the use of our approach in attempting to decipher the complex pathological processes leading to multiple sclerosis and other neurodegenerative diseases.  Further, we show that in clinicaly isolated syndrome (CIS), we could identify important molecules that could serve as an early diagnostic biomarker in MS.

Krishnakumar

Aug
13
Tue
2013
Delegate Talk: Inefficient NETosis: Cause for Predisposition to Recurrent Infections in Type 2 Diabetes @ Acharya Hall
Aug 13 @ 6:18 pm – 6:25 pm
Delegate Talk: Inefficient NETosis: Cause for Predisposition to Recurrent Infections in Type 2 Diabetes @ Acharya Hall | Vallikavu | Kerala | India

Manjunath Joshi, Apoorva Lad, Bharat Prasad Alevoor, Aswath Balakrishnan, Lingadakai Ramachandra and Kapaettu Satyamoorthy


 

Pathological conditions during Type 2 Diabetes (T2D) are associated with elevated risk for common community acquired infections due to poor glycemic control. Multiple studies have indicated specific defects in innate and adaptive immune function in diabetic subjects. Neutrophils play an important role in eliminating pathogens as an active constituent of innate immune system. Apart from canonically known phagocytosis mechanism, neutrophils are endowed with a unique ability to produce extracellular traps (NETs) to kill pathogens by expelling DNA coated with bactericidal proteins and histone. NETosis is stimulated by diverse bacteria and their products, fungi, protozoans, cytokines, phorbol esters and by activated platelets. Considering deregulation of metabolic and immune response pathways during pathological state of diabetes and NETosis as a potential mechanism for killing bacteria, we therefore, investigated whether hyperglycemic conditions modulate formation of neutrophil NETs and attempted to identify underlying immunoregulatory mechanisms. Freshly isolated neutrophils from normal individuals were cultured in absence or presence of high glucose (different concentrations) for 24 hours and activated with either LPS (2 mg/ml) or PMA (20 ng/ml) or IL-6 (20 ng/ml) for 3 hours. NETs were visualized and quantified by addition of DNA binding dye SYTOX green using fluorescence microscope and fluorimetry. NETs were quantified in Normal and diabetic subjects. Serum IL-6 levels were measured using ELISA technique. NETs bound elasatse were quantified in normal and diabetic subjects in presence or absence of DNase. Bacterial killing assays were performed upon infecting E.coli with activated neutrophils from normal and diabetic subjects. Microscopy and fluorimetry analysis suggested dramatic impairment in NETs formation under high glucose conditions. Extracellular DNA lattices formed in hyperglycemic conditions were short lived and unstable leading to rapid disintegration. Subsequent, time course experiments showed that NETs production was delayed in hyperglycemic conditions. To validate our findings more closely to clinical conditions, we investigated the neutrophil activation and NETs formation in diabetic patients. Upon stimulation with LPS for three hours, neutrophils from diabetic subjects responded weakly to LPS and lesser NETs were formed; whereas, neutrophils from normal individuals showed robust release of NETs. In few patients we found short and imperfect NETs in basal conditions suggesting constitutive activation of neutrophils in diabetic subjects. Interestingly, NETs bound elastase activity was reduced in diabetes subjects when compared to non-diabetic individuals, indicating a dysfunction of one of the important protein component of NETs during diabetes. Neutrophils from diabetic subjects released higher levels of IL-6 without any stimulation suggesting an existence of constitutively activated pro-inflammatory state. IL-6 induced NETs formation and was abrogated by high glucose. Weobserved that glycolysis inhibitor 2-DG resensitize the high glucose attenuated LPS and IL-6 induced NETs. a) NETs are influenced by glucose homeostasis, b) IL-6 as potent inducer of energy dependent NETs formation and c) hyperglycemia mimics a state of constitutively active pro-inflammatory condition in neutrophils leading to reduced response to external stimuli making diabetic subjects susceptible for infections.

Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall
Aug 13 @ 6:39 pm – 6:49 pm
Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall | Vallikavu | Kerala | India

Aswath Balakrishnan, Kapaettu Satyamoorthy and Manjunath B Joshi


Introduction
Insulin resistance is a hall mark of metabolic disorders such as diabetes. Reduced insulin response in vasculature leads to disruption of IR/Akt/eNOS signaling pathway resulting in vasoconstriction and subsequently to cardiovascular diseases. Recent studies have demonstrated that inflammatory regulator interleukin-6 (IL-6), as one of the potential mediators that can link chronic inflammation with insulin resistance. Accumulating evidences suggest a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases.

Materials and Methods
Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence of IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Using immunoblotting, cell lysates were stained for phosphor- and total Akt levels to measure insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 (at various concentrations) for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting.

Results
Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Aktser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of 5-methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL-6) as compared to untreated controls. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-α did not exhibit such changes.

Conclusion
Our study suggests: a) Chronic treatment of endothelial cells with IL-6 results in insulin resistance b) Neutrophil conditioned medium and IL-6 decreases methyl cytosine levels c) DNMT1 but not DNMT3a levels are reduced in cells treated with IL-6.