Río-Hortega's drawings revisited with fluorescent protein defines a cytoplasm-filled channel system of CNS myelin

A century ago this year, Pío del Río-Hortega (1921) coined the term ‘oligodendroglia’ for the ‘interfascicular glia’ with very few processes, launching an extensive discovery effort on his new cell type. One hundred years later, we review his original contributions to our understanding of the system of cytoplasmic channels within myelin in the context of what we observe today using light and electron microscopy of genetically encoded fluorescent reporters and immunostaining. We use the term myelinic channel system to describe the cytoplasm-delimited spaces associated with myelin; being the paranodal loops, inner and outer tongues, cytoplasm-filled spaces through compact myelin and further complex motifs associated to the sheath. Using a central nervous system myelinating cell culture model that contains all major neural cell types and produces compact myelin, we find that td-tomato fluorescent protein delineates the myelinic channel system in a manner reminiscent of the drawings of adult white matter by Río-Hortega, despite that he questioned whether some cytoplasmic figures he observed represented artefact. Together, these data lead us to propose a slightly revised model of the ‘unrolled’ sheath. Further, we show that the myelinic channel system, while relatively stable, can undergo subtle dynamic shape changes over days. Importantly, we capture an under-appreciated complexity of the myelinic channel system in mature myelin sheaths.     

Activation of myelin reactive T cells in multiple sclerosis: A possible role for T cell degeneracy?

While it is widely accepted that myelin reactive T cells are key players in the immunopathogenesis of multiple sclerosis (MS), the initial triggers that turn on these self-reactive T-cells remain to be determined. One mechanism, which is already text book knowledge for a decade, is molecular mimicry by which viral or microbial antigens are able to cross-activate T cells specific for myelin epitopes, the major target in MS pathology. Although proof of concept for this principle was given in animal model studies, evidence for such a mechanism in MS is limited. In this issue, Zhang et al. demonstrate an increased frequency of T cells that cross-react with a wide variety of antigens, a phenomenon termed as T cell degeneracy. While the role of these degenerate T cells in MS remains to be determined the authors now provide new elegant tools to study this T cell population, thus providing a starting point to better understand their function in MS.     

Early IFN-γ production together with decreased expression of TLR3 and TLR9 characterizes EAE development conditional on the presence of myelin

Experimental autoimmune encephalomyelitis (EAE) is a model for the study of multiple sclerosis, which is an inflammatory and demyelinating disease of the central nervous system (CNS). Despite increased efforts to elucidate the function of toll-like receptors (TLRs) in autoimmune diseases of the CNS, the relative contribution of other factors, including the immunomodulatory properties of TLR signaling, role of the innate response and the presence or absence of myelin peptides remain unclear. The aim was to evaluate TLR expression in the CNS during EAE development by investigating the expression of TLRs in the initial phase of EAE and establishing correlations with the modulation of inflammatory factors. Mice were subcutaneously immunized at the tail base with 100?μg of myelin oligodendrocyte glycoprotein peptide (MOG_35–55), emulsified in complete Freund’s adjuvant (CFA) supplemented with 400?μg of attenuated Mycobacterium tuberculosis H37RA. Pertussis toxin (300?ng per animal) was intraperitoneally injected on the day of immunization and 48?h later. Another group (MOG^?) received an equal emulsion of CFA and M. tuberculosis, without MOG_35–55, and the same protocol of Pertussis toxin. The immunized mice presented signs of disease with increased IFN-γ production and presence of NK cells on Day 2 postimmunization and reduced the expression of TLR-3 and TLR-9. In the spinal cord, CCL5 and CCL20 were higher in EAE. This study establishes a correlation between TLR-3 and TLR-9 expression with the development of EAE. In addition, evidence of a role for the myelin peptide in targeting the innate inflammatory response to the CNS is presented.     

Suppression of ongoing experimental allergic encephalomyelitis in DA rats by novel peptide drug,structural part of human myelin basic protein 46–62

Previously, we demonstrated that autoantibodies (AAb) in multiple sclerosis (MS) reveal site-specific binding and cleavage toward myelin basic protein (MBP) epitope library. We have found several fragments of MBP immunodominant in terms of AAb binding. Here, we applied these peptides to DA rats with induced protracted relapsing experimental allergic encephalomyelitis (EAE) most closely related to MS. DA rats with EAE induced by syngenic spinal cord homogenate in complete Freund's adjuvant were treated by nasal route with human MBP 46–62, 81–102, 124–139, 147–170, and Copaxone®. MBP 124–139 and 147–170 displayed only mild therapeutic effects but MBP 46–62 significantly reduced EAE, reflected by lower clinical scores and shorter EAE duration compared to controls.     

Myelin down-regulates myelin phagocytosis by microglia and macrophages through interactions between CD47 on myelin and SIRPα (signal regulatory protein-α) on phagocytes

Background  

Traumatic injury to axons produces breakdown of axons and myelin at the site of the lesion and then further distal to this where Wallerian degeneration develops. The rapid removal of degenerated myelin by phagocytosis is advantageous for repair since molecules in myelin impede regeneration of severed axons. Thus, revealing mechanisms that regulate myelin phagocytosis by macrophages and microglia is important. We hypothesize that myelin regulates its own phagocytosis by simultaneous activation and down-regulation of microglial and macrophage responses. Activation follows myelin binding to receptors that mediate its phagocytosis (e.g. complement receptor-3), which has been previously studied. Down-regulation, which we test here, follows binding of myelin CD47 to the immune inhibitory receptor SIRPα (signal regulatory protein-α) on macrophages and microglia.     

Amyloid β1-42 oligomer inhibits myelin sheet formation in vitro

Accumulating evidence indicates that white matter degeneration contributes to the neural disconnections that underlie Alzheimer's disease pathophysiology. Although this white matter degeneration is partly attributable to axonopathy associated with neuronal degeneration, amyloid β (Aβ) protein-mediated damage to oligodendrocytes could be another mechanism. To test this hypothesis, we studied effects of soluble Aβ in oligomeric form on survival and differentiation of cells of the oligodendroglial lineage using highly purified oligodendroglial cultures from rats at different developmental stages. Aβ oligomer at 10 μM or higher reduced survival of mature oligodendrocytes, whereas oligodendroglial progenitor cells (OPCs) were relatively resistant to the Aβ oligomer-mediated cytotoxicity. Further study revealed that Aβ oligomer even at 1 μM accelerated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formazan exocytosis in mature oligodendrocytes, and, more significantly, inhibited myelin sheet formation after induction of in vitro differentiation of OPCs. These results imply a novel pathogenetic mechanism underlying Aβ oligomer-mediated white matter degeneration, which could impair myelin maintenance and remyelination by adult OPCs, resulting in accumulating damage to myelinating axons thereby contributing to neural disconnections.     

Altered peptide ligands of myelin basic protein (MBP87–99) conjugated to reduced mannan modulate immune responses in mice

Mutations of peptides to generate altered peptide ligands, capable of switching immune responses from T helper 1 (Th1) to T helper 2 (Th2), are promising candidates for the immunotherapy of autoimmune diseases such as multiple sclerosis (MS). We synthesized two mutant peptides from myelin basic protein 87–99 (MBP_87–99), an immunodominant peptide epitope identified in MS. Mutations of residues K⁹¹ and P⁹⁶, known to be critical T-cell receptor (TCR) contact sites, resulted in the mutant peptides [R⁹¹, A⁹⁶]MBP_87–99 and [A⁹¹, A⁹⁶]MBP_87–99. Immunization of mice with these altered peptide ligands emulsified in complete Freund’s adjuvant induced both interferon-γ (IFN-γ) and interleukin-4 (IL-4) responses compared with only IFN-γ responses induced to the native MBP_87–99 peptide. It was of interest that [R⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan induced 70% less IFN-γ compared with the native MBP_87–99 peptide. However, [A⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan did not induce IFN-γ-secreting T cells, but elicited very high levels of interleukin-4 (IL-4). Furthermore, antibodies generated to [A⁹¹, A⁹⁶]MBP_87–99 peptide conjugated to reduced mannan did not cross-react with the native MBP_87–99 peptide. By molecular modelling of the mutant peptides in complex with major histocompatibility complex (MHC) class II, I-A^s, novel interactions were noted. It is clear that the double-mutant peptide analogue [A⁹¹, A⁹⁶]MBP_87–99 conjugated to reduced mannan is able to divert immune responses from Th1 to Th2 and is a promising mutant peptide analogue for use in studies investigating potential treatments for MS.     

Charcot–Marie–Tooth 1B caused by expansion of a familial myelin protein zero (MPZ) gene duplication

Charcot–Marie–Tooth (CMT) disease is a group of hereditary disorders affecting the motor and sensory nerves of the peripheral nervous system. CMT patterns of inheritance include dominant, recessive, and X-linked disorders. Charcot–Marie–Tooth disease, type 1B (CMT1B, OMIM 118200) is an autosomal dominant neuropathy caused by mutations in myelin protein zero (MPZ, OMIM 159440), a structural protein of peripheral myelin. Most causative MPZ mutations are missense sequence variants; however, recent clinical reports have described cases of CMT1B caused by increased dosage of the MPZ gene, with over-expression of the MPZ protein suspected to be causative of the disorder. We report an unusual case of early onset de novo CMT1B, caused by amplification of a familial, apparently benign, MPZ duplication.     

Studies of the Mechanism of Development of “Deprivation” Potentiation of Population Responses of Neurons in Field CA1 of Living Hippocampal Slices

Experiments on rat hippocampal slices were performed with testing of the synaptic connections of Schaffer collaterals and neurons in field CA1 to study the effects of interrupting low-frequency test stimulation (0.05 Hz) on the amplitude of population spikes. These studies demonstrated a correlation between the duration of pauses in stimulation (form 10 to 120 min) and increases in the amplitude of spikes (on average by 30–100% of baseline response magnitude). This deprivation potentiation was additive and could persist for long periods of time (testing was up to 1 h). Preliminary induction of long-term post-tetanic potentiation, which prevented the subsequent development of the late, but not the short-term phase, for 1–3 h, led to suppression of the development of deprivation potentiation after a 60-min pause in stimulation. Similar results were obtained in experiments using 20 M polymyxin B, which blocks protein kinase C and the PKC-dependent phase of long-term post-tetanic potentiation; this is evidence supporting the previously advanced hypothesis that the development of deprivation potentiation and the late, PKC-dependent phase of long-term post-tetanic potentiation share common mechanisms, associated with people phosphorylation.     

Mechanisms of recognition of the outlines of “vanishing” optotypes

The aim of the present work was to study the interaction between the optical properties of images of “ disappearing” optotypes and their recognition thresholds. The “disappearing” optotypes were figures with complex outlines and had a unique property-they were close to the threshold of recognition and observation, which increases the accuracy of measurement of visual acuity and the subjects’ attention to them. The recognition distances of “disappearing” optotypes were measured. A relationship was found between the recognition distance of “disappearing” optotypes and different optical density profiles on the one hand and the spatial and spatial frequency characteristics of the stimuli on the other. The decisive factor determining the threshold of recognition of optotypes in spatial frequency terms is its spatial frequency spectrum; that in spatial terms is the width of the black/white pair or black-white triad in the complex outline. Regardless of the shape of the optotype, one of the most important limiting factors was the concordance of this test with the scattering function of the subject’s eye optics. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 9, pp. 1080–1090, September, 2005.     

Effect of Cell–Cell Interactions on the Observable Strength of Adhesion of Sheets of Cells

Previous research in cellular adhesion has focused primarily on studying isolated cells under conditions where cells do not interact with each other. However, in vivo cells form sheets where both cell–substratum and cell–cell interactions contribute to the overall adhesive behavior. Our understanding of how cell–cell and cell–substratum interactions affect the overall process of cell adhesion in these situations is limited. To address this problem, we developed a systematic approach to evaluate how cell–cell and cell–substratum interactions affect the critical shear stress for detachment for semiconfluent and confluent sheets of cells. Our studies were based on subjecting cultures of adherent cells to a defined hydrodynamic flow in a radial-flow chamber with a gap height of 140 m. Using phase-contrast microscope imaging and analysis we measured shear-dependent patterns of detachment as a function of the extent of cell confluency. Our results show that the critical shear stress for detachment is maximum at intermediate extents of confluency of 10%–40%. These results have important implications for sodding vascular grafts and tissue engineering. © 1999 Biomedical Engineering Society. PAC99: 8718-h, 8719Rr     

Numerical Simulation of thePorous Structure of Biomaterials

Porous biomaterials are widely used as bone replacement materials because of thers high biocompatibility and osteoconductivity property. Understanding of their porous structure (i. e. geometrical and topological characteristic) and studying how to the body fluid flow through them are essential to investigate thed egradation behaviour at the surface-liquid interface. This research develops a numerical model to simulate the porous structure of biomaterials based on the stochastic approach in pore size distribution and interconnectivity.     

The Quantitative Stereologic Analysis of the Ultrastructure of Scirrhous Carcinoma of Breast

1.IntroductionInordertofindQutthebestwaytodiagnoseandtreatthecarcinomaofbreaSt,thestudyforpathology,andmorphometryorstereologyareverynecessary.ButthequantitativeanalysisoftheultrastructllreortheStere0l0gyaboutbreaStcarcinomahasnotyotbeenseenandnottobereported('x'x').Sowemeasured28morphologicalparametersofmit0chondrion,lysosome,roughendoplasmicreticulumandribosomeintencasesofscirrhouscarcinomaofbreastbystereologicmethods.Wehopedthatthisworkwouldpr0vides0mequantitativedaaforpathologyandearlydi…     

Investigation of the “valency” of antibodies by means of azoproteins

Summary The valency of antibodies was studied by the method of exhaustion of antisera against mono-and diazoproteins, and subsequent cross reactions both with the antibodies left over in the supernatant fluid of the serum and with the precipitating and nonprecipitating antibodies isolated from the precipitate.It was proved that the antibodies interact with the antigens as multivalent compounds.The valency determined with regard to the azoproteins is dependent upon the number of groups introduced.Thus, bivalent antibodies correspond to monoazoproteins and trivalent ones to diazoproteins.The valency of antibodies is, evidently, determined by the structural similarity of the heterologous and the immunizing antigens as well as by the less complete specific conformity between the individual structural peculiarities of the antigen and its antibody.From the Tashkent Pharmaceutical Institute (Director-Docent M. A. Azizov)Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov