An Approach to Teaching Object-Oriented Analysis and Design

This paper presents a syllabus that attempts to address the problem of teaching systems analysis and design in the changing world of today. In the first part of the paper, major issues and constraints that affect the development of a syllabus for this discipline are identified and analyzed. The second part of the paper focuses on the key points of a methodology constructed from traditional and object-oriented techniques, designed to satisfy the academic demands of the subject and reflect current practice, while providing students with a coherent and organized approach to systems analysis and design. Analysis of the outcomes and experience of implementing the syllabus provide the basis for conclusions and identification of possible areas for future research

Astroglial Cell-to-Cell Interaction with Autoreactive Immune Cells in Experimental Autoimmune Encephalomyelitis Involves P2X7 Receptor, 3-Integrin, and Connexin-43

In multiple sclerosis (MS), glial cells astrocytes interact with the autoreactive immune cells that attack the central nervous system (CNS), which causes and sustains neuroinflammation. However, little is known about the direct interaction between these cells when they are in close proximity in the inflamed CNS. By using an experimental autoimmune encephalomyelitis (EAE) model of MS, we previously found that in the proximity of autoreactive CNS-infiltrated immune cells (CNS-IICs), astrocytes respond with a rapid calcium increase that is mediated by the autocrine P2X7 receptor (P2X7R) activation. We now reveal that the mechanisms regulating this direct interaction of astrocytes and CNS-IICs involve the coupling between P2X7R, connexin-43, and β3-integrin. We found that P2X7R and astroglial connexin-43 interact and concentrate in the immediate proximity of the CNS-IICs in EAE. P2X7R also interacts with β3-integrin, and the block of astroglial αvβ3-integrin reduces the P2X7R-dependent calcium response of astrocytes upon encountering CNS-IICs. This interaction was dependent on astroglial mitochondrial activity, which regulated the ATP-driven P2X7R activation and facilitated the termination of the astrocytic calcium response evoked by CNS-IICs. By further defining the interactions between the CNS and the immune system, our findings provide a novel perspective toward expanding integrin-targeting therapeutic approaches for MS treatment by controlling the cell–cell interactions between astrocytes and CNS-IICs.casopis je u kategoriji M2

Novel Molecular Biomarkers at the Blood-Brain Barrier in ALS

Recently neuroinflammation has gained a particular focus as a key mechanism of ALS. Several studies in vivo as well as in vitro have nominated immunoglobulin G (IgG) isolated from ALS patients as an active contributor to disease onset and progression. We have shown that ALS IgG affects astroglial Ca2+ excitability and induces downstream activation of phosphatidylinositol 3-kinase. These studies were hampered by a lack of knowledge of the pathway of entry of immune factors in the CNS. Our MRI data revealed the blood-brain barrier BBB leakage and T cell infiltration into brain parenchyma in ALS G93A rats. Since astrocyte ensheathes blood vessel wall contributing to BBB stability and plays an important role in ALS pathogenesis, we have studied astrocytic membrane proteins water channel aquaporin-4 and the inwardly rectifying potassium channel. In this review, we will summarize data related to BBB disruption with particular emphasis on impaired function of astrocytes in ALS. We will discuss implication of membrane proteins expressed on astrocytic endfeet, aquaporin-4, and inwardly rectifying potassium channel in the pathology of ALS. In addition to ALS-specific IgGs, these membrane proteins are proposed as novel biomarkers of the disease

αVβ3-Integrin and mitochondria mediate astrocyte response to autoreactive immune cells

The astrocytic network maintains homeostasis in the central nervous system (CNS) through interactions with neighboring cells. In the CNS autoimmune disease, multiple sclerosis (MS), neuroinflammatory conditions modulate these cell-to-cell interactions. Our previous work revealed that the immune cells infiltrated into the CNS (CNS-IICs) of experimental autoimmune encemphalomyelitis (EAE) rat, an animal model of MS, rapidly alter the activity pattern of astrocytes by activating the glial P2X7 receptor (P2X7R). In the present study we further defined the mechanisms responsible for astrocytes’ activation in the presence of CNS-IICs. For this purpose, we used an in vitro experimental setup and monitored Ca2+ dynamics in Fluo-4-labeled cultured naïve astrocytes following brief bath application of CNS-IICs isolated from the spinal cord of the EAE rat. Our data indicate that the astroglial αvβ3-integrin is involved in the initial contact of astrocytes with CNS-IICs, since blocking αvβ3-integrin reduced the expected astrocytic Ca2+ response. Furthermore, blocking of mitochondrial Na+/Ca2+- and H+/Ca2+- exchangers in astrocytes promoted an augmentation of the intracellular Ca2+ increase and a higher ATP release after brief exposure to CNS-IICs, demonstrating that mitochondria regulate the astrocyte-CNS IICs cell-cell interaction. Overall, our study expands the understanding of astrocytes’ interaction with autoreactive immune cells that are present in their local environment in an autoimmune disease. This offers a new conceptual framework for considering direct astrocyte–immune cell interaction to design new strategies for therapy development in the treatment of MS

P2X7R, β3-integrin and Cx-43 mediate interaction between astrocytes and adjacent autoreactive immune cells

Astrocytes form a dense meshwork throughout the central nervous system (CNS) which qualifies them to perform interactive maintenance functions with neighboring cells. In neuroinflammation, this astroglial cell-to-cell interaction varies which can either promote or lessen pathological processes (1,2). In multiple sclerosis (MS), astrocytes engage in an interaction with immune cells which drives neurodegeneration by creating and sustaining an inflammatory CNS environment (3). Previously, we showed that CNS-infiltrated immune cells (CNS-IICs) in the experimental autoimmune encephalomyelitis (EAE) rat, rapidly alter the activity pattern of astrocytes by activating glial P2X7receptor (P2X7R)(4). In the present study, we aimed to identify the properties of astroglial P2X7R in EAE and to identify mechanisms responsible for astrocyte activation in the presence of CNS-IICs (CD4+T cells). In this respect, spinal cords from rats at the peak of EAE and age-matched healthy controls were isolated and protein expression of P2X7R and connexin-43 (Cx-43) were investigated. P2X7R protein expression was decreased in the lumbar spinal cord, while Cx-43 did not change. Next, we found that P2X7R and Cx-43 proteins interact in the lumbar spinal cord since both the monomer and the dimer Cx-43 co-immunoprecipitate with P2X7R. Even though the colocalization of P2X7R and Cx-43 was decreased in EAE compared to the control, the analysis of the distribution of astroglial P2X7R and Cx-43 and their colocalization in the radius of 20 μm from the infiltrated CD4+T cell center showed that astroglial P2X7R and Cx-43 are specifically associated and concentrated in the proximity of CNS-IICs in the EAE spinal cord. Subsequently, to achieve an unambiguous analysis of astrocyte-immune cell interaction, we monitored Ca2+dynamics in Fluo-4 labeled cultured naïve astrocytes following brief bath-application of CNS-IICs isolated and purified from spinal cords of EAE rats. Our data suggest that astroglial αvβ3-integrin acted upstream of P2X7R activation and is likely involved in establishing initial contact of astrocytes with CNS-IICs since astrocytic αvβ3-integrin block reduced the astrocytic Ca2+response to CNS-IIC application. Furthermore, astrocytes challenged with CGP31157 (blocker of mNCLX and HCX) exhibited a prolonged intracellular Ca2+elevation and higher ATP release after brief exposure to CNS-IICs, indicating a regulatory function of mitochondria on this intracellular astrocyte Ca2+response. Collectively these data describing integrin-relevant cellular mechanisms of astroglial P2X7R activation could help to expand integrin-inhibiting therapeutic approaches currently in use for MS treatment toward control ofastrocyte purine-based interaction with immune cells.kategorija M3

Approach for patch-clamping using an upright microscope with z-axis movable stage

We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping