Identification of Protein Networks Involved in the Disease Course of Experimental Autoimmune Encephalomyelitis, an Animal Model of Multiple Sclerosis

A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective therapies. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins involved in the development of inflammatory brain lesions, to help unravel underlying disease processes. Brainstem proteins were obtained from rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. We identified 75 unique proteins in 92 spots with a significant abundance difference between the experimental groups. To find disease-related networks, these regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). The analysis revealed that 70% of these proteins have been described to take part in neurological disease. Furthermore, some focus networks were created by IPA. These networks suggest an integrated regulation of the identified proteins with the addition of some putative regulators. Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1 (KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the identified proteins. Functional blocking of the KCNMA1 in macrophages was able to alter myelin phagocytosis, a disease mechanism highly involved in EAE and MS pathology. Quantitative analysis of differentially expressed brainstem proteins in an animal model of MS is a first step to identify disease-associated proteins and networks that warrant further research to study their actual contribution to disease pathology

Proteome Profiling in Murine Models of Multiple Sclerosis: Identification of Stage Specific Markers and Culprits for Tissue Damage

The identification of new biomarkers is of high interest for the prediction of the disease course and also for the identification of pathomechanisms in multiple sclerosis (MS). To specify markers of the chronic disease phase, we performed proteome profiling during the later phase of myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE, day 35 after immunization) as a model disease mimicking many aspects of secondary progressive MS. In comparison to healthy controls, high resolution 2 dimensional gel electrophoresis revealed a number of regulated proteins, among them glial fibrilary acidic protein (GFAP). Phase specific up-regulation of GFAP in chronic EAE was confirmed by western blotting and immunohistochemistry. Protein levels of GFAP were also increased in the cerebrospinal fluid of MS patients with specificity for the secondary progressive disease phase. In a next step, proteome profiling of an EAE model with enhanced degenerative mechanisms revealed regulation of alpha-internexin, syntaxin binding protein 1, annexin V and glutamate decarboxylase in the ciliary neurotrophic factor (CNTF) knockout mouse. The identification of these proteins implicate an increased apoptosis and enhanced axonal disintegration and correlate well the described pattern of tissue injury in CNTF −/− mice which involve oligodendrocyte (OL) apoptosis and axonal injury

Albumin and multiple sclerosis

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Leakage of the blood–brain barrier (BBB) is a common pathological feature in multiple sclerosis (MS). Following a breach of the BBB, albumin, the most abundant protein in plasma, gains access to CNS tissue where it is exposed to an inflammatory milieu and tissue damage, e.g., demyelination. Once in the CNS, albumin can participate in protective mechanisms. For example, due to its high concentration and molecular properties, albumin becomes a target for oxidation and nitration reactions. Furthermore, albumin binds metals and heme thereby limiting their ability to produce reactive oxygen and reactive nitrogen species. Albumin also has the potential to worsen disease. Similar to pathogenic processes that occur during epilepsy, extravasated albumin could induce the expression of proinflammatory cytokines and affect the ability of astrocytes to maintain potassium homeostasis thereby possibly making neurons more vulnerable to glutamate exicitotoxicity, which is thought to be a pathogenic mechanism in MS. The albumin quotient, albumin in cerebrospinal fluid (CSF)/albumin in serum, is used as a measure of blood-CSF barrier dysfunction in MS, but it may be inaccurate since albumin levels in the CSF can be influenced by multiple factors including: 1) albumin becomes proteolytically cleaved during disease, 2) extravasated albumin is taken up by macrophages, microglia, and astrocytes, and 3) the location of BBB damage affects the entry of extravasated albumin into ventricular CSF. A discussion of the roles that albumin performs during MS is put forth

P39

Microvesicles release by neoplastic cells were found in blood and carrying a large number of tumor distinctive molecules, like miRNA/mRNA, proteins. Considering extensive surface of blood cells we wonder if exosomesare circulated exclusively in plasma or could be associated with surface of blood cells. Materials and Methods: Microvesicles circulating in blood of healthy women (HW) and breast cancer patients (BCP) were studied. Blood was fractionated into plasma and cellular fractions; in order to release cell surface bound material, blood cells were treated as it was described for cell-surface-bound (CSB) cirDNA (Tamkovich, 2005).Exosomes were pelleted at 100,000 g from 0.1 μm filtered supernatants obtained by plasma and CSB eluates centrifugation at 17,000 g, resuspended in PBS and stored in aliquots at −80 °C. Size distribution of the microparticles was characterized by TEM (Jeol, Japan), anti CD-63, CD-24, CD-9 antibodies (BD Biosciences, USA) were used as exosome markers, protein concentration was measured by NanoOrange Protein Quantitation kit (Molecular Probes, USA), total RNA and miRNA were isolated by mirVana kit, 28S rRNA were quantified by RT-qPCR after reverse transcription with 6-mer random primers, miRNA using TaqMan Small RNA Assays kit (Applied Biosystems, USA). DNA was isolated using “DNA Isolation Kit” (BioSilicaLtd, Russia) and measured by TaqMan multiplex real-time PCR for LINE1 and α-satellite repeats (Bryzgunova, 2011). Results: TEM with immunogold labeling demonstrate presence of exosomes in 30–100 nm membrane-wrapped particles isolated from both plasma and CSB eluates. TEM, NanoOrange and 28S rRNA RT-qPCR data demonstrate that CSB exosomes constitutes 2/3 of total blood exosomes. Exosomes ranged 50–70 nm prevail in blood of BCP, whereas 30–50 nm exosomes in blood of HW. Exosomal DNA is less than 0.3% of cell-free blood DNA. RNA integrity and specific quantity checked by Bioanalyzer (Agilent Technologies, USA) do not differ in cell-free and CSB exosomes. Preliminary data demonstrate overpresentation of cancer-specific miRNA (miR-103, miR-191, miR-195) in exosomes bound with erythrocyte’s as compared with exosomes bound with leukocytes or circulating in plasma. Conclusion: Exosomal DNA obviously do not have any diagnostic value in contrast to RNA. CSB exosomes represent valuable source of material for small-invasive cancer diagnostics

Properties and applications of undecylprodigiosin and other bacterial prodigiosins

The growing demand to fulfill the needs of present-day medicine in terms of novel effective molecules has lead to reexamining some of the old and known bacterial secondary metabolites. Bacterial prodigiosins (prodiginines) have a long history of being re markable multipurpose compounds, best examined for their anticancer and antimalarial activities. Production of prodigiosin in the most common producer strain Serratia marcescens has been described in great detail. However, few reports have discussed the ecophysiological roles of these molecules in the producing strains, as well as their antibiotic and UV-protective properties. This review describes recent advances in the production process, biosynthesis, properties, and applications of bacterial prodigiosins. Special emphasis is put on undecylprodigiosin which has generally been a less studied member of the prodigiosin family. In addition, it has been suggested that proteins involved in undecylprodigiosin synthesis, RedG and RedH, could be a useful addition to the biocatalytic toolbox being able to mediate regio- and stereoselective oxidative cyclization. Judging by the number of recent references (216 for the 2007-2013 period), it has become clear that undecylprodigiosin and other bacterial prodigiosins still hold surprises in terms of valuable properties and applicative potential to medical and other industrial fields and that they still deserve continuing research curiosity